GPR156 variants and uses thereof

ABSTRACT

The disclosure provides nucleic acids, including cDNA, comprising alterations that encode aspartic acid at a position corresponding to position 533 of the human G protein-coupled receptor 156 protein (GPR156). The disclosure also provides isolated and recombinant human GPR156 protein variants that comprise an aspartic acid at a position corresponding to position 533. The change to aspartic acid, and the gene encoding this change, associate with unipolar depression. The disclosure also provides methods for determining whether a subject has or has a risk of developing unipolar depression, based on the identification of such alterations in the gene (DNA or RNA) encoding GPR156.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/367,973, filed on Jul. 28, 2016, the contents of which areincorporated by reference herein, in their entirety and for allpurposes.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named 18923800701SEQ, created on Jul. 28, 2017, with a sizeof 986 kilobytes. The Sequence Listing is incorporated by referenceherein.

FIELD

The disclosure relates generally to the field of genetics. Moreparticularly, the disclosure relates to gene alterations and polypeptidevariants in the G protein-coupled receptor 156 that associate with, forexample, unipolar depression and anxiety disorders.

BACKGROUND

Various references, including patents, patent applications, accessionnumbers, technical articles, and scholarly articles are cited throughoutthe specification. Each reference is incorporated by reference herein,in its entirety and for all purposes.

G protein-coupled receptors (GPCRs) are a large superfamily of cellsurface receptors that are characterized by 7 helical transmembranedomains, together with N-terminal extracellular and C-terminalintracellular domains (Cherezov V, et al., Science 318: 1258-1265(2007)). GPCRs are expressed in a variety of cell types, and participatein transducing extracellular signals across the cellular membrane andinto the cellular interior (Kobilka R, Biochim. Biophys. Acta 1768:794-807 (2007)). So important is the role that GPCRs play in biologythat in 2012, the Nobel Prize in Chemistry was awarded to two scientistswho identified how GPCRs function (Clark R, Proc. Natl. Acad. Sci. USA110: 5274-5275 (2013)).

GPR156 (G protein-coupled receptor 156), is a human gene which encodes aGPCR belonging to metabotropic glutamate receptor subfamily (Calver A,et al., Brain Res. Mol. Brain Res. 110: 305-307 (2003)). Other namesthat have been used to reference GPR156 in the literature are GABABL(GABAB-like) and PGR28 (Vassilatis D, Proc. Natl. Acad. Sci. USA 100:4903-4908 (2003)). In mice, GPR156 is referred to as Gpr156 or Gababl.Identifying new GPR156 variants would be helpful in the continued studyof the role of GPR156 in human physiology and its potential role indiseases. The present disclosure provides novel GPR156 variants thatwill aid in understanding the biology of GPR156, and will facilitate theidentification of potentially therapeutic agents that modulate GPR156and/or its cellular biological pathway.

SUMMARY

The present disclosure provides novel nucleic acid molecules (i.e.,genomic DNA, mRNA, and cDNA) encoding GPR156 variant polypeptides, andGPR156 variant polypeptides, that have been demonstrated herein to beassociated with a spectrum of mood disorders, such as depression, suchas unipolar depression, and anxiety disorders.

The present disclosure provides isolated nucleic acid moleculescomprising a nucleic acid sequence encoding a human GPR156 protein,wherein the protein comprises an aspartic acid at the positioncorresponding to position 533 according to SEQ ID NO:4, or thecomplement of the nucleic acid sequence.

The present disclosure also provides genomic DNA molecules comprising anucleic acid sequence encoding at least a portion of the human GPR156protein, wherein the portion comprises an aspartic acid at the positioncorresponding to position 533 according to SEQ ID NO:4, or thecomplement of the nucleic acid sequence.

The present disclosure also provides cDNA molecules comprising a nucleicacid sequence encoding at least a portion of the human GPR156 protein,wherein the portion comprises an aspartic acid at the positioncorresponding to position 533 according to SEQ ID NO:4, or thecomplement of the nucleic acid sequence.

The present disclosure also provides mRNA molecules comprising a nucleicacid sequence encoding at least a portion of the human GPR156 protein,wherein the portion comprises an aspartic acid at the positioncorresponding to position 533 according to SEQ ID NO:4, or thecomplement of the nucleic acid sequence.

The present disclosure also provides vectors comprising any of theisolated nucleic acid molecules disclosed herein.

The present disclosure also provides compositions comprising any of theisolated nucleic acid molecules or vectors disclosed herein and acarrier.

The present disclosure also provides host cells comprising any of theisolated nucleic acid molecules or vectors disclosed herein.

The present disclosure also provides isolated or recombinantpolypeptides comprising at least a portion of the human GPR156 protein,wherein the portion comprises an aspartic acid at the positioncorresponding to position 533 according to SEQ ID NO:4.

The present disclosure also provides compositions comprising any of theisolated or recombinant polypeptides disclosed herein and a carrier.

The present disclosure also provides a probe or a primer comprising anucleic acid sequence comprising at least about 5 nucleotides, whichhybridizes to a nucleic acid sequence encoding a human GPR156 proteincomprising an aspartic acid at the position corresponding to position at533 according to SEQ ID NO:4, or which hybridizes to the complement ofthe nucleic acid sequence encoding the human GPR156 protein comprisingthe aspartic acid at the position corresponding to position at 533according to SEQ ID NO:4.

The present disclosure also provides supports comprising a substrate towhich any of the probes disclosed herein hybridize.

The present disclosure also provides an allele-specific probe or primercomprising a nucleic acid sequence which is complementary to a nucleicacid sequence encoding a GPR156 protein, wherein the allele-specificprobe or primer comprises a nucleic acid sequence which is complementaryto the nucleic acid codon encoding an aspartic acid at the positioncorresponding to position 533 according to SEQ ID NO:4. In someembodiments, the allele-specific probe or primer specifically hybridizesto a nucleic acid sequence encoding a GPR156 protein comprising anaspartic acid at the position corresponding to position 533 according toSEQ ID NO:4, or to the complement thereof. The allele-specific probe orprimer does not hybridize to a nucleic acid sequence encoding a GPR156protein which does not comprise an aspartic acid at the positioncorresponding to position 533 according to SEQ ID NO:4.

The present disclosure also provides methods for diagnosing unipolardepression or detecting a risk of unipolar depression in a humansubject, comprising: detecting an alteration in a nucleic acid moleculeencoding a GPR156 protein obtained from the human subject, wherein thealteration encodes an aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4; and diagnosing the human subjectwith unipolar depression if the subject has one or more symptoms ofdepression, or diagnosing the human subject as at risk for unipolardepression if the subject does not have one or more symptoms ofdepression.

The present disclosure also provides methods for diagnosing an anxietydisorder or detecting a risk of an anxiety disorder in a human subject,comprising: detecting an alteration in a nucleic acid molecule encodinga GPR156 protein obtained from the human subject, wherein the alterationencodes an aspartic acid at the position corresponding to position 533according to SEQ ID NO:4; and diagnosing the human subject with ananxiety disorder if the subject has one or more symptoms of an anxietydisorder, or diagnosing the human subject as at risk for an anxietydisorder if the subject does not have one or more symptoms of an anxietydisorder.

The present disclosure also provides antidepressants for use in thetreatment of unipolar depression in a human subject determined tocomprise an alteration in a gene encoding the human GPR156 protein,wherein the alteration encodes an aspartic acid at the positioncorresponding to position 533 according to SEQ ID NO:4.

The present disclosure also provides antidepressants for use in thetreatment of an anxiety disorder in a human subject determined tocomprise an alteration in a gene encoding the human GPR156 protein,wherein the alteration encodes an aspartic acid at the positioncorresponding to position 533 according to SEQ ID NO:4.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the presentdisclosure.

FIG. 1 shows a vector map of mRor1ss.hGPR156 expression vector.

FIG. 2 shows a vector map of mRor1ss.hGPR156_E533D expression vector.

Additional advantages of the present disclosure will be set forth inpart in the description which follows, and in part will be apparent fromthe description, or can be learned by practice of the embodimentsdisclosed herein. The advantages of the present disclosure will berealized and attained by means of the elements and combinationsparticularly pointed out in the appended claims. It is to be understoodthat both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictiveof the embodiments, as claimed.

DESCRIPTION

Various terms relating to aspects of disclosure are used throughout thespecification and claims. Such terms are to be given their ordinarymeaning in the art, unless otherwise indicated. Other specificallydefined terms are to be construed in a manner consistent with thedefinition provided herein.

Unless otherwise expressly stated, it is in no way intended that anymethod or aspect set forth herein be construed as requiring that itssteps be performed in a specific order. Accordingly, where a methodclaim does not specifically state in the claims or descriptions that thesteps are to be limited to a specific order, it is in no way intendedthat an order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including matters of logic withrespect to arrangement of steps or operational flow, plain meaningderived from grammatical organization or punctuation, or the number ortype of aspects described in the specification.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

As used herein, the terms “subject” and “patient” are usedinterchangeably. A subject may include any animal, including mammals.Mammals include, without limitation, farm animals (e.g., horse, cow,pig), companion animals (e.g., dog, cat), laboratory animals (e.g.,mouse, rat, rabbits), and non-human primates. In some embodiments, thesubject is a human being.

As used herein, a “nucleic acid,” a “nucleic acid molecule,” a “nucleicacid sequence,” “polynucleotide,” or “oligonucleotide” can comprise apolymeric form of nucleotides of any length, may comprise DNA and/orRNA, and can be single-stranded, double-stranded, or multiple stranded.One strand of a nucleic acid also refers to its complement.

As used herein, the phrase “corresponding to” or grammatical variationsthereof when used in the context of the numbering of a given amino acidor nucleic acid sequence or position refers to the numbering of aspecified reference sequence when the given amino acid or nucleic acidsequence is compared to the reference sequence (e.g., with the referencesequence herein being the nucleic acid molecule or polypeptide of (wildtype or full length) GPR156). In other words, the residue (e.g., aminoacid or nucleotide) number or residue (e.g., amino acid or nucleotide)position of a given polymer is designated with respect to the referencesequence rather than by the actual numerical position of the residuewithin the given amino acid or nucleic acid sequence. For example, agiven amino acid sequence can be aligned to a reference sequence byintroducing gaps to optimize residue matches between the two sequences.In these cases, although the gaps are present, the numbering of theresidue in the given amino acid or nucleic acid sequence is made withrespect to the reference sequence to which it has been aligned.

For example, the phrase “GPR156 protein, wherein the protein comprisesan aspartic acid at the position corresponding to position 533 accordingto SEQ ID NO:4” means that, if the amino acid sequence of the GPR156protein is aligned to the sequence of SEQ ID NO:4, the GPR156 proteinhas an aspartic acid at the position that corresponds to position 533 ofSEQ ID NO:4. The same applies for the phrases “GPR156 protein, whereinthe protein comprises an aspartic acid at the position corresponding toposition 533 referring to SEQ ID NO:4” and “GPR156 protein, wherein theprotein comprises an aspartic acid at the position corresponding toposition 533 of SEQ ID NO:4.” Or, in other words, these phrases (e.g.the phrase “GPR156 protein, wherein the protein comprises an asparticacid at the position corresponding to position 533 according to SEQ IDNO:4”) refer to a GPR156 protein, which has an aspartic acid that ishomologous to the aspartic acid at position 533 of SEQ ID NO:4. Hereinsuch a protein is also referred to as “GPR156 protein with the E533Dmutation” or “GPR156 protein with the E533D variation.” In line withthis, the corresponding E533D variation is also referred to as “E533Dmutation (within the GPR156 protein)” or “E533D variation (within theGPR156 protein).”

As described above, a position within a GPR156 protein that correspondsto position 533 of SEQ ID NO:4 can easily be identified by performing asequence alignment between the given GPR156 protein and the amino acidsequence of SEQ ID NO:4. A variety of computational algorithms existthat can be used for performing a sequence alignment in order toidentify an amino acid position that corresponds to position 533 in SEQID NO:4. For example, by using the NCBI BLAST algorithm (Altschul et al.1997 Nucleic Acids Res. 25: 3389-3402) or CLUSTALW software (Sievers andHiggins 2014 Methods Mol. Biol. 1079: 105-116.) sequence alignments maybe performed. However, sequences can also be aligned manually.

It has been observed in accordance with the disclosure that certainvariations in GPR156 associate with a risk of developing mood disorders,such as unipolar depression and an anxiety disorder. In general, thefunction of this protein is poorly understood, with little publishedinformation about its biochemistry or its associated phenotypes. It isbelieved that no variants of the GPR156 gene or protein have any knownassociation with any mood or neurologic disorders in adult human beings.It is further believed that no variants of the GPR156 gene or proteinhave any known association with depression generally or unipolardepression or an anxiety disorder specifically in adult human beings. Arare variant in the GPR156 gene segregating with the phenotype ofunipolar depression in affected family members has been identified inaccordance with the disclosure. For example, a genetic alteration thatchanges the amino acid of position 533 in the human GPR156 protein(e.g., wild type SEQ ID NO:1) to aspartic acid has been observed toindicate that the human having such an alteration may develop unipolardepression. Altogether, the genetic analyses described hereinsurprisingly indicate that the GPR156 gene and, in particular, variantsin the GPR156 associate with increased susceptibility to unipolardepression and an anxiety disorder. Therefore, human subjects having GPR156 alterations that associate with unipolar depression or an anxietydisorder may be treated such that unipolar depression is inhibited, thesymptoms thereof are reduced, and/or development of symptoms isrepressed. Accordingly, the present disclosure provides isolated orrecombinant GPR156 variant genes, including cDNA and mRNA, as well asisolated or recombinant GPR156 variant polypeptides. Additionally, thedisclosure provides methods for leveraging the identification of suchvariants in subjects to identify or stratify risk in such subjects ofdeveloping unipolar depression or an anxiety disorder, or to diagnosesubjects as having unipolar depression or an anxiety disorder, such thatsubjects at risk or subjects with active disease may be treated.

The amino acid sequences for two full length wild type GPR156 proteinsare set forth in SEQ ID NO:1. Referring to SEQ ID NO:1, position 516 ofthe full length wild type GPR156 protein is either a glutamic acid or anaspartic acid. The amino acid sequence of another wild type GPR156protein is set forth in SEQ ID NO:2 (which contains a deletion of 4amino acids at positions corresponding to positions 198 to 201 of SEQ IDNO:1). Position 512 of this wild type GPR156 protein (referring to SEQID NO:2) is either a glutamic acid or an aspartic acid. In someembodiments, position 512 of this wild type GPR156 protein (referring toSEQ ID NO:2) is a glutamic acid. In some embodiments, position 512 ofthis wild type GPR156 protein (referring to SEQ ID NO:2) is an asparticacid. The amino acid sequence of a GPR156 protein comprising positions310 to 814 of SEQ ID NO:1 is set forth in SEQ ID NO:3. Positions 310 to814 of SEQ ID NO:1 define the cytoplasmic domain of GPR156. Position 207of this GPR156 protein (referring to SEQ ID NO:3) is either a glutamicacid or an aspartic acid. In some embodiments, position 207 of thisGPR156 protein (referring to SEQ ID NO:3) is a glutamic acid. In someembodiments, position 207 of this GPR156 protein (referring to SEQ IDNO:3) is an aspartic acid.

The present disclosure provides nucleic acid molecules encoding GPR156variant proteins that associate with unipolar depression or an anxietydisorder. For example, the present disclosure provides isolated nucleicacid molecules comprising a nucleic acid sequence encoding a humanGPR156 protein, wherein the protein comprises an aspartic acid at theposition corresponding to position 533 (E533D) according to SEQ ID NO:4,or the complement of the nucleic acid sequence. In some embodiments, theposition corresponding to position 516 of SEQ ID NO:4 is a glutamicacid. In some embodiments, the position corresponding to position 516 ofSEQ ID NO:4 is an aspartic acid.

An isoform of GPR156 exists, wherein four amino acids in the N-terminalto central region of the protein are deleted. The amino acid sequence ofthis GPR156 isoform is shown in SEQ ID NO:5. In this GPR156 isoformposition 529 corresponds to position 533 according to SEQ ID NO:4. Insome embodiments, the isolated nucleic acid molecules comprise a nucleicacid sequence encoding a human GPR156 protein, wherein the proteincomprises an aspartic acid at the position corresponding to position 529(E529D) according to SEQ ID NO:5, or the complement of the nucleic acidsequence. In some embodiments, position 512 of SEQ ID NO:5 is a glutamicacid. In some embodiments, the position corresponding to position 512 ofSEQ ID NO:5 is an aspartic acid.

In some embodiments, the nucleic acid molecule comprises or consists ofa nucleic acid sequence that encodes a human GPR156 protein having anamino acid sequence that has at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% sequence identity to SEQ ID NO:4, wherein the GPR156protein comprises an aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4, or the complement of the nucleicacid sequence. In some embodiments, the position corresponding toposition 516 of this nucleic acid molecule (referring to SEQ ID NO:4) isa glutamic acid. In some embodiments, the position corresponding toposition 516 of this nucleic acid molecule (referring to SEQ ID NO:4) isan aspartic acid. Herein, if reference is made to percent sequenceidentity, the higher percentages of sequence identity are preferred overthe lower ones.

In some embodiments, the nucleic acid molecule comprises or consists ofa nucleic acid sequence that encodes a human GPR156 protein having anamino acid sequence that has at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% sequence identity to SEQ ID NO:5, wherein the GPR156protein comprises an aspartic acid at the position corresponding toposition 529 according to SEQ ID NO:5, or the complement of the nucleicacid sequence. In some embodiments, the position corresponding toposition 512 of this GPR156 protein (referring to SEQ ID NO:5) is aglutamic acid. In some embodiments, the position corresponding toposition 512 of this GPR156 protein (referring to SEQ ID NO:5) is anaspartic acid.

In some embodiments, the nucleic acid sequence comprises DNA and theaspartic acid at the position corresponding to position 533 is encodedby the codon GAT or GAC. In some embodiments, the nucleic acid sequencecomprises RNA and the aspartic acid at the position corresponding toposition 533 is encoded by the codon GAU or GAC in the RNA molecule. Insome embodiments, the aspartic acid at the position corresponding toposition 533 is encoded by the codon GAT. In some embodiments, theGPR156 protein encoded by the nucleic acid sequence comprises the aminoacid sequence of SEQ ID NO:4 or SEQ ID NO:5.

The nucleic acid sequences for two wild type GPR156 genomic DNAs (i.e.,alternate alleles) are set forth in SEQ ID NO:7 (see, “n” at position117,166, which can be a, g, t, or c). Positions 117,164 to 117,166 ofthese wild type GPR156 genomic DNAs (referring to SEQ ID NO:7) encodeeither a glutamic acid (via the codon GAA or GAG) or an aspartic acid(via the codon GAT or GAC). Positions 117,215 to 117,217 of these wildtype GPR156 genomic DNAs (referring to SEQ ID NO:7) encode a glutamicacid (via the codon GAA or GAG).

The nucleic acid sequence of another wild type GPR156 genomic DNA is setforth in SEQ ID NO:8 (which contains a deletion of 12 nucleotides atpositions corresponding to positions 98,428 to 98,439 of SEQ ID NO:7).Positions 117,152 to 117,154 of this wild type GPR156 genomic DNA(referring to SEQ ID NO:8) encode either a glutamic acid (via the codonGAA or GAG) or an aspartic acid (via the codon GAT or GAC). Positions117,203 to 117,205 of this wild type GPR156 genomic DNA (referring toSEQ ID NO:8) encode a glutamic acid (via the codon GAA or GAG).

In some embodiments, the nucleic acid molecule is genomic DNA. In someembodiments, the genomic DNA comprises or consists of a nucleic acidsequence of SEQ ID NO:9 (where positions 117,215 to 117,217 are GAT,thereby encoding an aspartic acid) or SEQ ID NO:10 (where positions117,215 to 117,217 are GAC, thereby encoding an aspartic acid), or anucleic acid sequence that has at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99% sequence identity to SEQ ID NO:9 (and comprisesthe GAT codon at the position corresponding to the position encoding theaspartic acid at position 533 according to SEQ ID NO:4) or SEQ ID NO:10(and comprises the GAC codon at the position corresponding to theposition encoding the aspartic acid at position 533 according to SEQ IDNO:4). In some embodiments, positions 117,164 to 117,166 of these GPR156genomic DNAs (referring to SEQ ID NO:9 and SEQ ID NO:10) encode aglutamic acid (via the codon GAA or GAG). In some embodiments, thepositions corresponding to positions 117,164 to 117,166 of these GPR156genomic DNAs (referring to SEQ ID NO:9 and SEQ ID NO:10) encode anaspartic acid (via the codon GAT or GAC).

In some embodiments, the genomic DNA comprises or consists of a nucleicacid sequence of SEQ ID NO:11 or SEQ ID NO:12, each of which contains adeletion of 12 nucleotides at positions corresponding to positions98,428 to 98,439 of SEQ ID NO:9 and SEQ ID NO:10, respectively. In someembodiments, the genomic DNA comprises or consists of a nucleic acidsequence set forth in SEQ ID NO:11, where positions 117,203 to 117,205are GAT, thereby encoding an aspartic acid. In some embodiments, thegenomic DNA comprises or consists of a nucleic acid sequence set forthin SEQ ID NO:12,where positions 117,203 to 117,205 are GAC, therebyencoding an aspartic acid. In some embodiments, the genomic DNAcomprises or consists of a nucleic acid sequence that has at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity to SEQID NO:11 (and comprises the GAT codon at the position corresponding tothe position encoding the aspartic acid at position 529 according to SEQID NO:5) or SEQ ID NO:12 (and comprises the GAC codon at the positioncorresponding to the position encoding the aspartic acid at position 529according to SEQ ID NO:5). In some embodiments, the positionscorresponding to positions 117,152 to 117,154 of these GPR156 genomicDNAs (referring to SEQ ID NO:11 and SEQ ID NO:12) encode a glutamic acid(via the codon GAA or GAG). In some embodiments, the positionscorresponding to positions 117,152 to 117,154 of these GPR156 genomicDNAs (referring to SEQ ID NO:11 and SEQ ID NO:12) encode an asparticacid (via the codon GAT or GAC).

In some embodiments, the isolated nucleic acid molecules comprise lessthan the entire genomic DNA sequence. In some embodiments, the isolatednucleic acid molecules comprise or consist of at least about 15, atleast about 20, at least about 25, at least about 30, at least about 35,at least about 40, at least about 45, at least about 50, at least about60, at least about 70, at least about 80, at least about 90, at leastabout 100, at least about 200, at least about 300, at least about 400,at least about 500, at least about 600, at least about 700, at leastabout 800, at least about 900, at least about 1000, at least about 2000,at least about 3000, at least about 4000, at least about 5000, at leastabout 6000, at least about 7000, at least about 8000, at least about9000, at least about 10000, at least about 11000, at least about 12000,at least about 13000, at least about 14000, at least about 15000, atleast about 16000, at least about 17000, at least about 18000, at leastabout 19000, or at least about 20000 contiguous nucleotides of SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments,the isolated nucleic acid molecules comprise or consist of at leastabout 1000 to at least about 2000 contiguous nucleotides of SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

In some embodiments, the isolated nucleic acid molecules comprise orconsist of at least about 15, at least about 20, at least about 25, atleast about 30, at least about 35, at least about 40, at least about 45,at least about 50, at least about 60, at least about 70, at least about80, at least about 90, at least about 100, at least about 200, at leastabout 300, at least about 400, at least about 500, at least about 600,at least about 700, at least about 800, at least about 900, at leastabout 1000, at least about 1000, at least about 1100, at least about1200, at least about 1300, at least about 1400, at least about 1500, atleast about 1600, at least about 1700, at least about 1800, at leastabout 1900, at least about 2000, at least about 2100, at least about2200, at least about 2300, or at least about 2400 contiguous nucleotidesof SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In someembodiments, such contiguous nucleotides may be combined with othernucleic acid molecules of contiguous nucleotides to produce the cDNAmolecules described herein.

Such isolated nucleic acid molecules can be used, for example, toexpress variant GPR156 mRNAs and proteins or as exogenous donorsequences. It is understood that gene sequences within a population canvary due to polymorphisms, such as SNPs. The examples provided hereinare only exemplary sequences, and other sequences are also possible.

In some embodiments, the isolated nucleic acid molecules comprise avariant GPR156 minigene, in which one or more nonessential segmentsencoding SEQ ID NO:4 or SEQ ID NO:5 have been deleted with respect to acorresponding wild type GPR156 gene. In some embodiments, the deletednonessential segments comprise one or more intron sequences. In someembodiments, the GPR156 minigene has at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or 100% sequence identity to a portion of SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, wherein theminigene comprises aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4.

The nucleic acid sequences for two wild type GPR156 cDNAs are set forthin SEQ ID NO:13. Positions 1546 to 1548 of these wild type GPR156 cDNAs(referring to SEQ ID NO:13) encode either a glutamic acid (via the codonGAA or GAG) or an aspartic acid (via the codon GAT or GAC). Positions1597 to 1599 of these wild type GPR156 cDNAs (referring to SEQ ID NO:13)encode a glutamic acid (via the codon GAA or GAG).

The nucleic acid sequence of another wild type GPR156 cDNA is set forthin SEQ ID NO:14 (which contains a deletion of 12 nucleotides atpositions corresponding to positions 592-603 of SEQ ID NO:13). Positions1534 to 1536 of this wild type GPR156 cDNA (referring to SEQ ID NO:14)encode either a glutamic acid (via the codon GAA or GAG) or an asparticacid (via the codon GAT or GAC). Positions 1585 to 1587 of this wildtype GPR156 cDNA (referring to SEQ ID NO:14) encode a glutamic acid (viathe codon GAA or GAG).

The nucleic acid sequence of a GPR156 cDNA encoding positions 310 to 814of SEQ ID NO:4 is set forth in SEQ ID NO:15. This sequence correspondsto the cytoplasmic domain of GPR156. Positions 619 to 621 of this GPR156cDNA (referring to SEQ ID NO:15) encode either a glutamic acid (via thecodon GAA or GAG) or an aspartic acid (via the codon GAT or GAC).Positions 670 to 672 of this GPR156 cDNA (referring to SEQ ID NO:15)encode a glutamic acid (via the codon GAA or GAG).

The present disclosure also provides cDNA molecules. The presentdisclosure provides a cDNA molecule comprising a nucleic acid sequenceencoding at least a portion of the human GPR156 protein, wherein theportion comprises an aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4. In some embodiments, the cDNAmolecule comprises a nucleic acid sequence encoding at least a portionof the human GPR156 protein, wherein the portion comprises an asparticacid at the position corresponding to position 529 according to SEQ IDNO:5.

In some embodiments, the cDNA encodes a full length human GPR156 proteincomprising an aspartic acid at the position corresponding to position533 according to SEQ ID NO:4. In some embodiments, the cDNA comprises orconsists of a nucleic acid sequence that encodes a polypeptide having anamino acid sequence that has at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% sequence identity to SEQ ID NO:4, wherein the GPR156protein comprises an aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4.

In some embodiments, the cDNA encodes a human GPR156 protein comprisingan aspartic acid at the position corresponding to position 529 accordingto SEQ ID NO:5. In some embodiments, the cDNA comprises or consists of anucleic acid sequence that encodes a polypeptide having an amino acidsequence that has at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity to SEQ ID NO:5, wherein the GPR156 proteincomprises an aspartic acid at the position corresponding to position 529according to SEQ ID NO:5.

In some embodiments, the cDNA encodes the portion of the GPR156 proteincorresponding to positions 310 to 814 of SEQ ID NO:4. In someembodiments, the cDNA comprises or consists of a nucleic acid sequencethat encodes a polypeptide having an amino acid sequence that has atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99% sequenceidentity to the portion of the GPR156 protein corresponding to positions310 to 814 of SEQ ID NO:4, wherein the polypeptide comprises an asparticacid at the position corresponding to position 533 according to SEQ IDNO:4.

In some embodiments, the cDNA encodes the portion of the GPR156 proteincorresponding to positions 310 to 814 of SEQ ID NO:5. In someembodiments, the cDNA comprises or consists of a nucleic acid sequencethat encodes a polypeptide having an amino acid sequence that has atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99% sequenceidentity to the portion of the GPR156 protein corresponding to positions310 to 814 of SEQ ID NO:5, wherein the polypeptide comprises an asparticacid at the position corresponding to position 533 according to SEQ IDNO:5.

In some embodiments, the cDNA comprises or consists of a nucleic acidsequence of SEQ ID NO:16, where positions 1597 to 1599 are GAT, therebyencoding an aspartic acid. In some embodiments, the cDNA comprises orconsists of a nucleic acid sequence that has at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% sequence identity to SEQ IDNO:16, and comprises the GAT codon at the position corresponding to theposition encoding the aspartic acid at position 533 according to SEQ IDNO:4). In some embodiments, the positions corresponding to positions1546 to 1548 of the GPR156 cDNA (referring to SEQ ID NO:16) encode aglutamic acid. In some embodiments, the positions corresponding topositions 1546 to 1548 of the GPR156 cDNA (referring to SEQ ID NO:16)encode an aspartic acid. In some embodiments, the cDNA comprises athymine at the position corresponding to position 1599 of the GPR156cDNA (referring to SEQ ID NO:16). This thymine corresponds to the thirdposition of the codon encoding the aspartic acid at the pocitioncorresponding to position 533 according to SEQ ID NO:4).

In some embodiments, the cDNA comprises or consists of a nucleic acidsequence of SEQ ID NO:17, where positions 1597 to 1599 are GAC, therebyencoding an aspartic acid. In some embodiments, the cDNA comprises orconsists of a nucleic acid sequence that has at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% sequence identity to SEQ IDNO:17, and comprises the GAC codon at the position corresponding to theposition encoding the aspartic acid at position 533 according to SEQ IDNO:4). In some embodiments, the positions corresponding to positions1546 to 1548 of the GPR156 cDNA (referring to SEQ ID NO:17) encode aglutamic acid. In some embodiments, positions 1546 to 1548 of the GPR156cDNA (referring to SEQ ID NO:17) encode an aspartic acid. In someembodiments, the cDNA comprises a cytosine at the position correspondingto position 1599 of the GPR156 cDNA (referring to SEQ ID NO:17). Thiscytosine corresponds to the third position of the codon encoding theaspartic acid at the position corresponding to position 533 according toSEQ ID NO:4).

In some embodiments, the cDNA comprises or consists of a nucleic acidsequence of SEQ ID NO:18, which contains a deletion of 12 nucleotides atpositions corresponding to positions 592 to 603 of SEQ ID NO:16. In someembodiments, the cDNA comprises or consists of a nucleic acid sequenceset forth in SEQ ID NO:18, where positions 1585 to 1587 are GAT, therebyencoding an aspartic acid. In some embodiments, the cDNA comprises orconsists of a nucleic acid sequence that has at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% sequence identity to SEQ ID NO:18(and comprises the GAT codon at the position corresponding to theposition encoding the aspartic acid at the position corresponding toposition 529 according to SEQ ID NO:5). In some embodiments, thepositions corresponding to positions 1534 to 1536 of this GPR156 cDNA(referring to SEQ ID NO:18) encode a glutamic acid (via the codon GAA orGAG). In some embodiments, the positions corresponding to positions 1534to 1536 of this GPR156 cDNA (referring to SEQ ID NO:18) encode anaspartic acid (via the codon GAT or GAC). In some embodiments, the cDNAcomprises a thymine at the position corresponding to position 1587 ofthe GPR156 cDNA (referring to SEQ ID NO:18). This thymine corresponds tothe third position of the codon encoding the aspartic acid at theposition corresponding to position 529 according to SEQ ID NO:5).

In some embodiments, the cDNA comprises or consists of a nucleic acidsequence of SEQ ID NO:19, which contains a deletion of 12 nucleotides atpositions corresponding to positions 592 to 603 of SEQ ID NO:17. In someembodiments, the cDNA comprises or consists of a nucleic acid sequenceset forth in SEQ ID NO:19, where positions 1585 to 1587 are GAC, therebyencoding an aspartic acid. In some embodiments, the cDNA comprises orconsists of a nucleic acid sequence that has at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% sequence identity to SEQ ID NO:19(and comprises the GAC codon at the position corresponding to theposition encoding the aspartic acid at position 529 according to SEQ IDNO:5). In some embodiments, the positions corresponding to positions1534 to 1536 of this GPR156 cDNA (referring to SEQ ID NO:19) encode aglutamic acid (via the codon GAA or GAG). In some embodiments, thepositions corresponding to positions 1534 to 1536 of this GPR156 cDNA(referring to SEQ ID NO:19) encode an aspartic acid (via the codon GATor GAC). In some embodiments, the cDNA comprises a cytosine at theposition corresponding to position 1587 of the cDNA (referring to SEQ IDNO:19). This cytosine corresponds to the third position of the codonencoding the aspartic acid at the position corresponding to position 529according to SEQ ID NO:5).

The cytoplasmic domain of GPR156 is encoded by positions 310 to 814 ofGPR156. In some embodiments, the cDNA encodes positions 310 to 814 ofSEQ ID NO:4. In some embodiments, the nucleic acid sequence of this cDNAis set forth in SEQ ID NO:20, wherein positions 670 to 672 encode anaspartic acid (via the codon GAT). In some embodiments, the cDNAcomprises or consists of a nucleic acid sequence that has at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity to SEQID NO:20, and comprises the GAT codon at the position corresponding tothe position encoding the aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4. In some embodiments, thepositions corresponding to positions 619 to 621 of the GPR156 cDNA(referring to SEQ ID NO:20) encode a glutamic acid. In some embodiments,the positions corresponding to positions 619 to 621 of the GPR156 cDNA(referring to SEQ ID NO:20) encode an aspartic acid. In someembodiments, the cDNA comprises a thymine at the position correspondingto position 672 of the cDNA (referring to SEQ ID NO:20). This thyminecorresponds to the third position of the codon encoding the asparticacid at the position corresponding to position 533 according to SEQ IDNO:4).

In some embodiments, the nucleic acid sequence of the cDNA encodingpositions 310 to 814 of SEQ ID NO:4 is set forth in SEQ ID NO:21,wherein positions 670 to 672 encode an aspartic acid (via the codonGAC). In some embodiments, the cDNA comprises or consists of a nucleicacid sequence that has at least about 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity to SEQ ID NO:21, and comprises the GAC codonat the position corresponding to the position encoding the aspartic acidat the position corresponding to position 533 according to SEQ ID NO:4.In some embodiments, the positions corresponding to positions 619 to 621of the GPR156 cDNA (referring to SEQ ID NO:21) encode a glutamic acid.In some embodiments, the positions corresponding to positions 619 to 621of the GPR156 cDNA (referring to SEQ ID NO:21) encode an aspartic acid.In some embodiments, the cDNA comprises a cytosine at the positioncorresponding to position 672 of the cDNA (referring to SEQ ID NO:21).This cytosine corresponds to the third position of the codon encodingthe aspartic acid at the position corresponding to position 533according to SEQ ID NO:4).

In some embodiments, the isolated nucleic acid molecules comprise anucleic acid sequence comprising positions 1521 through 1680 of any ofthe cDNA or mRNA molecules disclosed herein, having a thymine at theposition corresponding to position 1599.

In some embodiments, the cDNA molecules comprise less than the entirecDNA sequence of GPR156. In some embodiments, the cDNA moleculescomprise or consist of at least about 5, at least about 8, at leastabout 10, at least about 12, at least about 15, at least about 20, atleast about 25, at least about 30, at least about 35, at least about 40,at least about 45, at least about 50, at least about 60, at least about70, at least about 80, at least about 90, at least about 100, at leastabout 200, at least about 300, at least about 400, at least about 500,at least about 600, at least about 700, at least about 800, at leastabout 900, at least about 1000, at least about 1100, at least about1200, at least about 1300, at least about 1400, at least about 1500, atleast about 1600, at least about 1700, at least about 1800, at leastabout 1900, at least about 2000, at least about 2100, at least about2200, at least about 2300, or at least about 2400 contiguous nucleotidesof SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,or SEQ ID NO:21. In some embodiments, the cDNA molecule comprises orconsists of at least about 1000 to at least about 2000 contiguousnucleotides of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, or SEQ ID NO:21. In this regard, the longer cDNA moleculesare preferred over the shorter ones. In some embodiments, the cDNAmolecules comprise or consist of at least about 15, at least about 20,at least about 25, at least about 30, at least about 35, at least about40, at least about 45, at least about 50, at least about 60, at leastabout 70, at least about 80, at least about 90, at least about 100, atleast about 200, at least about 300, at least about 400, or at leastabout 500 contiguous nucleotides of SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21. In this regard, thelonger cDNA molecules are preferred over the shorter ones. In someembodiments, such cDNA molecules include the codon that encodes theaspartic acid at the position that corresponds to position 533 accordingto SEQ ID NO:4, or includes a thymine or cysteine at the positioncorresponding to position 1599 according to SEQ ID NO:16 or SEQ IDNO:17, respectively.

The nucleic acid sequences for two wild type GPR156 mRNAs are set forthin SEQ ID NO:22. Positions 1546 to 1548 of these wild type GPR156 mRNAs(referring to SEQ ID NO:22) encode either a glutamic acid (via the codonGAA or GAG) or an aspartic acid (via the codon GAU or GAC). Positions1597 to 1599 of these wild type GPR156 mRNAs (referring to SEQ ID NO:22)encode a glutamic acid (via the codon GAA or GAG).

The nucleic acid sequence of another wild type GPR156 mRNA is set forthin SEQ ID NO:23 (which contains a deletion of 12 nucleotides atpositions corresponding to positions 592 to 603 of SEQ ID NO:22).Positions 1534 to 1536 of this wild type GPR156 mRNA (referring to SEQID NO:23) encode either a glutamic acid (via the codon GAA or GAG) or anaspartic acid (via the codon GAU or GAC). Positions 1585 to 1587 of thiswild type GPR156 mRNA (referring to SEQ ID NO:23) encode a glutamic acid(via the codon GAA or GAG).

The nucleic acid sequence of a GPR156 mRNA encoding positions 310 to 814of SEQ ID NO:4 is set forth in SEQ ID NO:24. Positions 619 to 621 ofthis GPR156 mRNA (referring to SEQ ID NO:24) encode either a glutamicacid (via the codon GAA or GAG) or an aspartic acid (via the codon GAUor GAC). Positions 670 to 672 of this GPR156 mRNA (referring to SEQ IDNO:24) encode a glutamic acid (via the codon GAA or GAG).

The present disclosure also provides mRNA molecules. The presentdisclosure provides mRNA molecules comprising a nucleic acid sequenceencoding at least a portion of the human GPR156 protein, wherein theportion comprises an aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4. In some embodiments, the mRNAmolecules comprise a nucleic acid sequence encoding at least a portionof the human GPR156 protein, wherein the portion comprises an asparticacid at the position corresponding to position 529 according to SEQ IDNO:5.

In some embodiments, the mRNA encodes a full length human GPR156 proteincomprising an aspartic acid at the position corresponding to position533 according to SEQ ID NO:4. In some embodiments, the mRNA comprises orconsists of a nucleic acid sequence that encodes a polypeptide having anamino acid sequence that has at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% sequence identity to SEQ ID NO:4, wherein the GPR156protein comprises an aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4.

In some embodiments, the mRNA encodes a human GPR156 protein comprisingan aspartic acid at the position corresponding to position 529 accordingto SEQ ID NO:5. In some embodiments, the mRNA comprises or consists of anucleic acid sequence that encodes a polypeptide having an amino acidsequence that has at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity to SEQ ID NO:5, wherein the GPR156 proteincomprises an aspartic acid at the position corresponding to position 529according to SEQ ID NO:5.

In some embodiments, the mRNA encodes the portion of the GPR156 proteincorresponding to positions 310 to 814 of SEQ ID NO:4. In someembodiments, the mRNA comprises or consists of a nucleic acid sequencethat encodes a polypeptide having an amino acid sequence that has atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99% sequenceidentity to the portion of the GPR156 protein corresponding to positions310 to 814 of SEQ ID NO:4, wherein the polypeptide comprises asparticacid at the position corresponding to position 533 according to SEQ IDNO:4.

In some embodiments, the mRNA encodes the portion of the GPR156 proteincorresponding to positions 310 to 814 of SEQ ID NO:5. In someembodiments, the mRNA comprises or consists of a nucleic acid sequencethat encodes a polypeptide having an amino acid sequence that has atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99% sequenceidentity to the portion of the GPR156 protein corresponding to positions310 to 814 of SEQ ID NO:5, wherein the polypeptide comprises asparticacid at the position corresponding to position 533 according to SEQ IDNO:5.

In some embodiments, the mRNA comprises or consists of a nucleic acidsequence of SEQ ID NO:25, where positions 1597 to 1599 are GAU, therebyencoding an aspartic acid. In some embodiments, the mRNA comprises orconsists of a nucleic acid sequence that has at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% sequence identity to SEQ IDNO:25, and comprises the GAU codon at the position corresponding to theposition encoding the aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4). In some embodiments, thepositions corresponding to positions 1546 to 1548 of the GPR156 mRNA(referring to SEQ ID NO:25) encode a glutamic acid. In some embodiments,the positions corresponding to positions 1546 to 1548 of the GPR156 mRNA(referring to SEQ ID NO:25) encode an aspartic acid. In someembodiments, the mRNA comprises a uracil at the position correspondingto position 1599 of the GPR156 mRNA (referring to SEQ ID NO:25). Thisuracil corresponds to the third position of the codon encoding theaspartic acid at the position corresponding to position 533 according toSEQ ID NO:4).

In some embodiments, the mRNA comprises or consists of a nucleic acidsequence of SEQ ID NO:26, where positions 1597 to 1599 are GAC, therebyencoding an aspartic acid. In some embodiments, the mRNA comprises orconsists of a nucleic acid sequence that has at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% sequence identity to SEQ IDNO:26, and comprises the GAC codon at the position corresponding to theposition encoding the aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4). In some embodiments, thepositions corresponding to positions 1546 to 1548 of the GPR156 mRNA(referring to SEQ ID NO:26) encode a glutamic acid. In some embodiments,the positions corresponding to positions 1546 to 1548 of the GPR156 mRNA(referring to SEQ ID NO:26) encode an aspartic acid. In someembodiments, the mRNA comprises a cytosine at the position correspondingto position 1599 of the GPR156 mRNA (referring to SEQ ID NO:26). Thiscytosine corresponds to the third position of the codon encoding theaspartic acid at the position corresponding to position 533 according toSEQ ID NO:4).

In some embodiments, the mRNA comprises or consists of a nucleic acidsequence of SEQ ID NO:27, which contains a deletion of 12 nucleotides atpositions corresponding to positions 592 to 603 of SEQ ID NO:25. In someembodiments, the mRNA comprises or consists of a nucleic acid sequenceset forth in SEQ ID NO:27, where positions 1585 to 1587 are GAU, therebyencoding an aspartic acid. In some embodiments, the mRNA comprises orconsists of a nucleic acid sequence that has at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% sequence identity to SEQ ID NO:27(and comprises the GAU codon at the position corresponding to theposition encoding the aspartic acid at the position corresponding toposition 529 according to SEQ ID NO:5). In some embodiments, thepositions corresponding to positions 1534 to 1536 of this GPR156 mRNA(referring to SEQ ID NO:27) encode a glutamic acid (via the codon GAA orGAG). In some embodiments, the positions corresponding to positions 1534to 1536 of this GPR156 mRNA (referring to SEQ ID NO:27) encode anaspartic acid (via the codon GAU or GAC). In some embodiments, the mRNAcomprises a uracil at the position corresponding to position 1587 of theGPR156 mRNA (referring to SEQ ID NO:27). This uracil corresponds to thethird position of the codon encoding the aspartic acid at the positioncorresponding to position 529 according to SEQ ID NO:5).

In some embodiments, the mRNA comprises or consists of a nucleic acidsequence of SEQ ID NO:28, which contains a deletion of 12 nucleotides atpositions corresponding to positions 592 to 603 of SEQ ID NO:26. In someembodiments, the mRNA comprises or consists of a nucleic acid sequenceset forth in SEQ ID NO:28, where positions 1585 to 1587 are GAC, therebyencoding an aspartic acid. In some embodiments, the mRNA comprises orconsists of a nucleic acid sequence that has at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% sequence identity to SEQ ID NO:28(and comprises the GAC codon at the position corresponding to theposition encoding the aspartic acid at position 529 according to SEQ IDNO:5). In some embodiments, the positions corresponding to positions1534 to 1536 of this GPR156 mRNA (referring to SEQ ID NO:28) encode aglutamic acid (via the codon GAA or GAG). In some embodiments, thepositions corresponding to positions 1534 to 1536 of this GPR156 mRNA(referring to SEQ ID NO:28) encode an aspartic acid (via the codon GAUor GAC). In some embodiments, the mRNA comprises a cytosine at theposition corresponding to position 1587 of the GPR156 mRNA (referring toSEQ ID NO:28). This cytosine corresponds to the third position of thecodon encoding the aspartic acid at the position corresponding toposition 529 according to SEQ ID NO:5).

In some embodiments, the mRNA encodes positions 310 to 814 of SEQ IDNO:4. In some embodiments, the nucleic acid sequence of this mRNA is setforth in SEQ ID NO:29, wherein positions 670 to 672 encode an asparticacid (via the codon GAU). In some embodiments, the mRNA comprises orconsists of a nucleic acid sequence that has at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% sequence identity to SEQ IDNO:29, and comprises the GAU codon at the position corresponding to theposition encoding the aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4. In some embodiments, thepositions corresponding to positions 619 to 621 of the GPR156 mRNA(referring to SEQ ID NO:29) encode a glutamic acid. In some embodiments,the positions corresponding to positions 619 to 621 of the GPR156 mRNA(referring to SEQ ID NO:29) encode an aspartic acid. In someembodiments, the mRNA comprises a uracil at the position correspondingto position 672 of the GPR156 mRNA (referring to SEQ ID NO:29). Thisuracil corresponds to the third position of the codon encoding theaspartic acid at the position corresponding to position 533 according toSEQ ID NO:4).

In some embodiments, the nucleic acid sequence of the mRNA encodingpositions 310 to 814 of SEQ ID NO:4 is set forth in SEQ ID NO:30,wherein positions 670 to 672 encode an aspartic acid (via the codonGAC). In some embodiments, the mRNA comprises or consists of a nucleicacid sequence that has at least about 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity to SEQ ID NO:30, and comprises the GAC codonat the position corresponding to the position encoding the aspartic acidat the position corresponding to position 533 according to SEQ ID NO:4.In some embodiments, the positions corresponding to positions 619 to 621of the GPR156 mRNA (referring to SEQ ID NO:30) encode a glutamic acid.In some embodiments, the positions corresponding to positions 619 to 621of the GPR156 mRNA (referring to SEQ ID NO:30) encode an aspartic acid.In some embodiments, the mRNA comprises a cytosine at the positioncorresponding to position 672 of the GPR156 mRNA (referring to SEQ IDNO:30). This cytosine corresponds to the third position of the codonencoding the aspartic acid at the position corresponding to position 533according to SEQ ID NO:4).

In some embodiments, the isolated nucleic acid molecules comprising anucleic acid sequence comprising positions 1521 through 1680 of any mRNAmolecules disclosed herein, having a uracil at the positioncorresponding to position 1599.

In some embodiments, the isolated nucleic acid molecule comprises lessnucleotides than the entire GPR156 mRNA sequence. In some embodiments,the isolated nucleic acid molecules comprise or consist of at leastabout 5, at least about 8, at least about 10, at least about 12, atleast about 15, at least about 20, at least about 25, at least about 30,at least about 35, at least about 40, at least about 45, at least about50, at least about 60, at least about 70, at least about 80, at leastabout 90, at least about 100, at least about 200, at least about 300, atleast about 400, at least about 500, at least about 600, at least about700, at least about 800, at least about 900, at least about 1000, atleast about 1100, at least about 1200, at least about 1300, at leastabout 1400, at least about 1500, at least about 1600, at least about1700, at least about 1800, at least about 1900, at least about 2000, atleast about 2100, at least about 2200, at least about 2300, or at leastabout 2400 contiguous nucleotides of SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. In some embodiments,the isolated nucleic acid molecules comprise or consist of at leastabout 1000 to at least about 2000 contiguous nucleotides of SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ IDNO:30. In this regard, the longer mRNA molecules are preferred over theshorter ones. In some embodiments, the isolated nucleic acid moleculescomprise or consist of at least about 15, at least about 20, at leastabout 25, at least about 30, at least about 35, at least about 40, atleast about 45, at least about 50, at least about 60, at least about 70,at least about 80, at least about 90, at least about 100, at least about200, at least about 300, at least about 400, at least about 500, atleast about 600, at least about 700, at least about 800, at least about900, or at least about 1000 contiguous nucleotides of SEQ ID NO:25, SEQID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. Inthis regard, the longer mRNA molecules are preferred over the shorterones. In some embodiments, such mRNA molecules include the codon thatencodes the aspartic acid at the position corresponding to position 533according to SEQ ID NO:4, or includes a uracil or cysteine correspondingto position 1599 of SEQ ID NO:25 or SEQ ID NO:26, respectively.

The present disclosure also provides isolated nucleic acid moleculesthat hybridize to variant GPR156 genomic DNAs (such as SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, or SEQ ID NO:12), variant GPR156 minigenes,variant GPR156 cDNAs (such as SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21), and/or variant GPR156mRNAs (such as SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:29, or SEQ ID NO:30). In some embodiments, such isolatednucleic acid molecules comprise or consist of at least about 5, at leastabout 8, at least about 10, at least about 11, at least about 12, atleast about 13, at least about 14, at least about 15, at least about 16,at least about 17, at least about 18, at least about 19, at least about20, at least about 21, at least about 22, at least about 23, at leastabout 24, at least about 25, at least about 30, at least about 35, atleast about 40, at least about 45, at least about 50, at least about 55,at least about 60, at least about 65, at least about 70, at least about75, at least about 80, at least about 85, at least about 90, at leastabout 95, at least about 100, at least about 200, at least about 300, atleast about 400, at least about 500, at least about 600, at least about700, at least about 800, at least about 900, at least about 1000, atleast about 2000, at least about 3000, at least about 4000, at leastabout 5000, at least about 6000, at least about 7000, at least about8000, at least about 9000, at least about 10000, at least about 11000,at least about 12000, at least about 13000, at least about 14000, atleast about 15000, at least about 16000, at least about 17000, at leastabout 18000, at least about 19000, or at least about 20000 nucleotides.In some embodiments, the isolated nucleic acid molecule comprises orconsists of at least 15 nucleotides. In some embodiments, the isolatednucleic acid molecule comprises or consists of at least 15 nucleotidesto at least about 35 nucleotides. In some embodiments, such isolatednucleic acid molecules hybridize to variant GPR156 genomic DNAs (such asSEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12), variantGPR156 minigenes, variant GPR156 cDNAs (such as SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21),and/or variant GPR156 mRNAs (such as SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30) under stringentconditions. Such nucleic acid molecules may be used, for example, asprobes, as primers, or as allele-specific primers as described orexemplified herein.

In some embodiments, the isolated nucleic acid molecules hybridize to atleast about 15 contiguous nucleotides of a nucleic acid molecule that isat least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or 100%identical to variant GPR156 genomic DNAs (such as SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, or SEQ ID NO:12), variant GPR156 minigenes, variantGPR156 cDNAs (such as SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, or SEQ ID NO:21), and/or variant GPR156 mRNAs (suchas SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,or SEQ ID NO:30). In some embodiments, the isolated nucleic acidmolecules comprise or consist of from about 15 to about 100 nucleotides,or from about 15 to about 35 nucleotides. In some embodiments, theisolated nucleic acid molecules comprise or consist of from about 15 toabout 100 nucleotides. In some embodiments, the isolated nucleic acidmolecules comprise or consist of rom about 15 to about 35 nucleotides.

In some embodiments, any of the nucleic acid molecules, genomic DNAmolecules, cDNA molecules, or mRNA molecules disclosed herein can bepurified, e.g., are at least about 90% pure. In some embodiments, any ofthe nucleic acid molecules, genomic DNA molecules, cDNA molecules, ormRNA molecules disclosed herein can be purified, e.g., are at leastabout 95% pure. In some embodiments, any of the nucleic acid molecules,genomic DNA molecules, cDNA molecules, or mRNA molecules disclosedherein can be purified, e.g., are at least about 99% pure. Purificationis according to the hands of a human being, with human-made purificationtechniques.

The present disclosure also provides fragments of any of the isolatednucleic acid molecules, genomic DNA molecules, cDNA molecules, or mRNAmolecules disclosed herein. In some embodiments, the fragments compriseor consist of at least about 5, at least about 8, at least about 10, atleast about 11, at least about 12, at least about 13, at least about 14,at least about 15, at least about 16, at least about 17, at least about18, at least about 19, at least about 20, at least about 21, at leastabout 22, at least about 23, at least about 24, at least about 25, atleast about 30, at least about 35, at least about 40, at least about 45,at least about 50, at least about 55, at least about 60, at least about65, at least about 70, at least about 75, at least about 80, at leastabout 85, at least about 90, at least about 95, or at least about 100contiguous residues of any of the nucleic acid sequences disclosedherein, or any complement thereof. In this regard, the longer fragmentsare preferred over the shorter ones. In some embodiments, the fragmentscomprise or consist of at least about 5, at least about 8, at leastabout 10, at least about 11, at least about 12, at least about 13, atleast about 14, at least about 15, at least about 16, at least about 17,at least about 18, at least about 19, at least about 20, at least about21, at least about 22, at least about 23, at least about 24, at leastabout 25, at least about 30, at least about 35, at least about 40, atleast about 45, or at least about 50 contiguous residues. In thisregard, the longer fragments are preferred over the shorter ones. Insome embodiments, the fragments comprise or consist of at least about20, at least about 25, at least about 30, or at least about 35contiguous residues. In some embodiments, the fragments comprise orconsist of at least about 20 contiguous residues. In some embodiments,the fragments comprise or consist of at least about 25 contiguousresidues. In some embodiments, the fragments comprise or consist of atleast about 30 contiguous residues. In some embodiments, the fragmentscomprise or consist of at least about 35 contiguous residues. It isenvisaged that the fragments comprise of consist of the portion of thenucleic acid molecule that encodes the aspartic acid at the positioncorresponding to position 533 of the protein having the amino acidsequence according to SEQ ID NO:4, or that encodes the aspartic acid atthe position corresponding to position 529 of the protein having theamino acid sequence according to SEQ ID NO:5. Such fragments may beused, for example, as probes, as primers, or as allele-specific primersas described or exemplified herein.

The present disclosure also provides probes and primers. The probe orprimer of the present disclosure have a nucleic acid sequence thatspecifically hybridizes to any of the nucleic acid molecules disclosedherein, or the complement thereof. In some embodiments, the probe orprimer specifically hybridizes to any of the nucleic acid moleculesdisclosed herein under stringent conditions. The present disclosure alsoprovides nucleic acid molecules having nucleic acid sequences thathybridize under moderate conditions to any of the nucleic acid moleculesdisclosed herein, or the complement thereof. A probe or primer accordingto the disclosure preferably encompasses the nucleic acid codon whichencodes the E533D variation within the GPR156 protein, or the complementthereof. Thus, in a preferred embodiment, the disclosure providesallele-specific primers which are defined herein above and below in moredetail.

A probe or primer according to the disclosure may be used to detect theE533D variation within the nucleic acid sequence encoding the GPR156protein (e.g., according to SEQ ID NO:4) and/or the E529D mutationwithin the nucleic acid molecule encoding the isoform of GPR156 (e.g.,according to SEQ ID NO:5). For example, a primer according to thedisclosure may be used to amplify GPR156 or a fragment thereofcomprising the E533D variation. A primer according to the disclosure mayalso be used to amplify a GPR156 isoform (such as the isoform having theamino acid sequence of SEQ ID NO:5) or a fragment thereof comprising theE529D mutation. Exemplified primers according to the disclosure areshown in Table 1.

The disclosure also provides a pair of primers comprising one of theprimers described above. By using such a pair of primers lengthpolymorphisms within GPR156 may also be detected. For example, a pair ofprimers may be used to distinguish the nucleic acid molecule encodingthe canonical GPR156 protein (e.g. of SEQ ID NO:4) from the nucleic acidmolecule encoding the GPR156 isoform which lacks four amino acids in theN-terminal to central region of the GPR156 protein (e.g. of SEQ IDNO:5). More specifically, if the positions where the primers hybridizeflank the positions of the four amino acids that are lacking in theGPR156 isoform, then the amplified fragment which corresponds to thecanonical GRP156 protein is longer as compared to the amplified fragmentwhich corresponds to the GPR156 isoform.

The nucleic acid molecules disclosed herein can comprise a nucleic acidsequence of a naturally occurring GPR156 gene, cDNA, or mRNA transcript,or can comprise a non-naturally occurring sequence. In some embodiments,the naturally occurring sequence can differ from the non-naturallyoccurring sequence due to synonymous mutations or mutations that do notaffect the encoded GPR156 polypeptide. For example, the sequence can beidentical with the exception of synonymous mutations or mutations thatdo not affect the encoded GPR156 polypeptide. A synonymous mutation orsubstitution is the substitution of one nucleotide for another in anexon of a gene coding for a protein such that the produced amino acidsequence is not modified. This is possible because of the degeneracy ofthe genetic code, with some amino acids being coded for by more than onethree-base pair codon. Synonymous substitutions are used, for example,in the process of codon optimization. The nucleic acid moleculesdisclosed herein can be codon optimized.

Also provided herein are functional polynucleotides that can interactwith the disclosed nucleic acid molecules. Functional polynucleotidesare nucleic acid molecules that have a specific function, such asbinding a target molecule or catalyzing a specific reaction. Examples offunctional polynucleotides include, but are not limited to, antisensemolecules, aptamers, ribozymes, triplex forming molecules, and externalguide sequences. The functional polynucleotides can act as effectors,inhibitors, modulators, and stimulators of a specific activity possessedby a target molecule, or the functional polynucleotides can possess a denovo activity independent of any other molecules.

Antisense molecules are designed to interact with a target nucleic acidmolecule through either canonical or non-canonical base pairing. Theinteraction of the antisense molecule and the target molecule isdesigned to promote the destruction of the target molecule through, forexample, RNase-H-mediated RNA-DNA hybrid degradation. Alternately, theantisense molecule is designed to interrupt a processing function thatnormally would take place on the target molecule, such as transcriptionor replication. Antisense molecules can be designed based on thesequence of the target molecule. Numerous methods for optimization ofantisense efficiency by identifying the most accessible regions of thetarget molecule exist. Exemplary methods include, but are not limitedto, in vitro selection experiments and DNA modification studies usingDMS and DEPC. Antisense molecules generally bind the target moleculewith a dissociation constant (k_(d)) less than or equal to about 10⁻⁶,less than or equal to about 10⁻⁸, less than or equal to about 10⁻¹⁰, orless than or equal to about 10⁻¹². A representative sample of methodsand techniques which aid in the design and use of antisense moleculescan be found in the following non-limiting list of U.S. Pat. Nos.5,135,917; 5,294,533; 5,627,158; 5,641,754; 5,691,317; 5,780,607;5,786,138; 5,849,903; 5,856,103; 5,919,772; 5,955,590; 5,990,088;5,994,320; 5,998,602; 6,005,095; 6,007,995; 6,013,522; 6,017,898;6,018,042; 6,025,198; 6,033,910; 6,040,296; 6,046,004; 6,046,319; and6,057,437. Examples of antisense molecules include, but are not limitedto, antisense RNAs, small interfering RNAs (siRNAs), and short hairpinRNAs (shRNAs).

The isolated nucleic acid molecules disclosed herein can comprise RNA,DNA, or both RNA and DNA. The isolated nucleic acid molecules can alsobe linked or fused to a heterologous nucleic acid sequence, such as in avector, or a heterologous label. For example, the isolated nucleic acidmolecules disclosed herein can be in a vector or exogenous donorsequence comprising the isolated nucleic acid molecule and aheterologous nucleic acid sequence. The isolated nucleic acid moleculescan also be linked or fused to a heterologous label, such as afluorescent label. Other examples of labels are disclosed elsewhereherein.

The label can be directly detectable (e.g., fluorophore) or indirectlydetectable (e.g., hapten, enzyme, or fluorophore quencher). Such labelscan be detectable by spectroscopic, photochemical, biochemical,immunochemical, or chemical means. Such labels include, for example,radiolabels that can be measured with radiation-counting devices;pigments, dyes or other chromogens that can be visually observed ormeasured with a spectrophotometer; spin labels that can be measured witha spin label analyzer; and fluorescent labels (e.g., fluorophores),where the output signal is generated by the excitation of a suitablemolecular adduct and that can be visualized by excitation with lightthat is absorbed by the dye or can be measured with standardfluorometers or imaging systems. The label can also be, for example, achemiluminescent substance, where the output signal is generated bychemical modification of the signal compound; a metal-containingsubstance; or an enzyme, where there occurs an enzyme-dependentsecondary generation of signal, such as the formation of a coloredproduct from a colorless substrate. The term “label” can also refer to a“tag” or hapten that can bind selectively to a conjugated molecule suchthat the conjugated molecule, when added subsequently along with asubstrate, is used to generate a detectable signal. For example, one canuse biotin as a tag and then use an avidin or streptavidin conjugate ofhorseradish peroxidate (HRP) to bind to the tag, and then use acalorimetric substrate (e.g., tetramethylbenzidine (TMB)) or afluorogenic substrate to detect the presence of HRP. Exemplary labelsthat can be used as tags to facilitate purification include, but are notlimited to, myc, HA, FLAG or 3XFLAG, 6XHis or polyhistidine,glutathione-S-transferase (GST), maltose binding protein, an epitopetag, or the Fc portion of immunoglobulin. Numerous labels are known andinclude, for example, particles, fluorophores, haptens, enzymes andtheir calorimetric, fluorogenic and chemiluminescent substrates andother labels.

The disclosed nucleic acid molecules can comprise, for example,nucleotides or non-natural or modified nucleotides, such as nucleotideanalogs or nucleotide substitutes. Such nucleotides include a nucleotidethat contains a modified base, sugar, or phosphate group, or thatincorporates a non-natural moiety in its structure. Examples ofnon-natural nucleotides include, but are not limited to,dideoxynucleotides, biotinylated, aminated, deaminated, alkylated,benzylated, and fluorophor-labeled nucleotides.

The nucleic acid molecules disclosed herein can also comprise one ormore nucleotide analogs or substitutions. A nucleotide analog is anucleotide which contains a modification to either the base, sugar, orphosphate moieties. Modifications to the base moiety include, but arenot limited to, natural and synthetic modifications of A, C, G, and T/U,as well as different purine or pyrimidine bases such as, for example,pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and2-aminoadenin-9-yl. Modified bases include, but are not limited to,5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Certain nucleotideanalogs such as, for example, 5-substituted pyrimidines,6-azapyrimidines, and N-2, N-6 and O-6 substituted purines including,but not limited to, 2-aminopropyladenine, 5-propynyluracil,5-propynylcytosine, and 5-methylcytosine can increase the stability ofduplex formation. Often, base modifications can be combined with, forexample, a sugar modification, such as 2′-O-methoxyethyl, to achieveunique properties such as increased duplex stability.

Nucleotide analogs can also include modifications of the sugar moiety.Modifications to the sugar moiety include, but are not limited to,natural modifications of the ribose and deoxy ribose as well assynthetic modifications. Sugar modifications include, but are notlimited to, the following modifications at the 2′ position: OH; F; O-,S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl may besubstituted or unsubstituted C₁₋₁₀alkyl or C₂₋₁₀alkenyl, andC₂₋₁₀alkynyl. Exemplary 2′ sugar modifications also include, but are notlimited to, −O[(CH₂)_(n)O]_(m),CH₃, —O(CH₂)_(n)OCH₃, —O(CH₂)_(n)NH₂,—O(CH₂)_(n)CH₃, −O(CH₂)_(n)—ONH₂, —O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10.

Other modifications at the 2′ position include, but are not limited to,C₁₋₁₀alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl orO-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂,NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, an RNA cleaving group, a reportergroup, an intercalator, a group for improving the pharmacokineticproperties of an oligonucleotide, or a group for improving thepharmacodynamic properties of an oligonucleotide, and other substituentshaving similar properties. Similar modifications may also be made atother positions on the sugar, particularly the 3′ position of the sugaron the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides andthe 5′ position of 5′ terminal nucleotide. Modified sugars can alsoinclude those that contain modifications at the bridging ring oxygen,such as CH₂ and S. Nucleotide sugar analogs can also have sugarmimetics, such as cyclobutyl moieties in place of the pentofuranosylsugar.

Nucleotide analogs can also be modified at the phosphate moiety.Modified phosphate moieties include, but are not limited to, those thatcan be modified so that the linkage between two nucleotides contains aphosphorothioate, chiral phosphorothioate, phosphorodithioate,phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates including 3′-alkylene phosphonate and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates. These phosphate or modified phosphate linkage betweentwo nucleotides can be through a 3′-5′ linkage or a 2′-5′ linkage, andthe linkage can contain inverted polarity such as 3′-5′ to 5′-3′ or2′-5′ to 5′-2′. Various salts, mixed salts, and free acid forms are alsoincluded.

Nucleotide substitutes include molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes includemolecules that will recognize nucleic acids in a Watson-Crick orHoogsteen manner, but which are linked together through a moiety otherthan a phosphate moiety. Nucleotide substitutes are able to conform to adouble helix type structure when interacting with the appropriate targetnucleic acid.

Nucleotide substitutes also include nucleotides or nucleotide analogsthat have had the phosphate moiety or sugar moieties replaced. In someembodiments, nucleotide substitutes may not contain a standardphosphorus atom. Substitutes for the phosphate can be, for example,short chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These include those having morpholino linkages (formed in part from thesugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxideand sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S, and CH₂ component parts.

It is also understood in a nucleotide substitute that both the sugar andthe phosphate moieties of the nucleotide can be replaced by, forexample, an amide type linkage (aminoethylglycine) (PNA).

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance, for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include, for example, lipid moietiessuch as a cholesterol moiety, cholic acid, a thioether such ashexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain such asdodecandiol or undecyl residues, a phospholipid such asdi-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, adamantane acetic acid, a palmityl moiety, oran octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

The present disclosure also provides vectors comprising any one or moreof the nucleic acid molecules disclosed herein. In some embodiments, thevectors comprise any one or more of the nucleic acid molecules disclosedherein and a heterologous nucleic acid. The vectors can be viral ornonviral vectors capable of transporting a nucleic acid molecule. Insome embodiments, the vector is a plasmid or cosmid (e.g., a circulardouble-stranded DNA into which additional DNA segments can be ligated).In some embodiments, the vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. In some embodiments,the vector can autonomously replicate in a host cell into which it isintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). In some embodiments, thevector (e.g., non-episomal mammalian vectors) can be integrated into thegenome of a host cell upon introduction into the host cell and therebyare replicated along with the host genome. Moreover, particular vectorscan direct the expression of genes to which they are operatively linked.Such vectors are referred to herein as “recombinant expression vectors”or “expression vectors.” Such vectors can also be targeting vectors(i.e., exogenous donor sequences).

In some embodiments, the proteins encoded by the various geneticvariants disclosed herein are expressed by inserting nucleic acidmolecules encoding the disclosed genetic variants into expressionvectors, such that the genes are operatively linked to expressioncontrol sequences, such as transcriptional and translational controlsequences. Expression vectors include, but are not limited to, plasmids,cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV),plant viruses such as cauliflower mosaic virus and tobacco mosaic virus,yeast artificial chromosomes (YACs), Epstein-Barr (EBV)-derivedepisomes, and other expression vectors known in the art. In someembodiments, nucleic acid molecules comprising the disclosed geneticvariants can be ligated into a vector such that transcriptional andtranslational control sequences within the vector serve their intendedfunction of regulating the transcription and translation of the geneticvariant. The expression vector and expression control sequences arechosen to be compatible with the expression host cell used. Nucleic acidsequences comprising the disclosed genetic variants can be inserted intoseparate vectors or into the same expression vector as the variantgenetic information. A nucleic acid sequence comprising the disclosedgenetic variants can be inserted into the expression vector by standardmethods (e.g., ligation of complementary restriction sites on thenucleic acid comprising the disclosed genetic variants and vector, orblunt end ligation if no restriction sites are present).

In addition to a nucleic acid sequence comprising the disclosed geneticvariants, the recombinant expression vectors can carry regulatorysequences that control the expression of the genetic variant in a hostcell. The design of the expression vector, including the selection ofregulatory sequences can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,and so forth. Desired regulatory sequences for mammalian host cellexpression can include, for example, viral elements that direct highlevels of protein expression in mammalian cells, such as promotersand/or enhancers derived from retroviral LTRs, cytomegalovirus (CMV)(such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as theSV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdMLP)), polyoma and strong mammalian promoters such as nativeimmunoglobulin and actin promoters. Methods of expressing polypeptidesin bacterial cells or fungal cells (e.g., yeast cells) are also wellknown.

A promoter can be, for example, a constitutively active promoter, aconditional promoter, an inducible promoter, a temporally restrictedpromoter (e.g., a developmentally regulated promoter), or a spatiallyrestricted promoter (e.g., a cell-specific or tissue-specific promoter).Examples of promoters can be found, for example, in WO 2013/176772.

Examples of inducible promoters include, for example, chemicallyregulated promoters and physically-regulated promoters. Chemicallyregulated promoters include, for example, alcohol-regulated promoters(e.g., an alcohol dehydrogenase (alcA) gene promoter),tetracycline-regulated promoters (e.g., a tetracycline-responsivepromoter, a tetracycline operator sequence (tetO), a tet-On promoter, ora tet-Off promoter), steroid regulated promoters (e.g., a ratglucocorticoid receptor, a promoter of an estrogen receptor, or apromoter of an ecdysone receptor), or metal-regulated promoters (e.g., ametalloprotein promoter). Physically regulated promoters include, forexample temperature-regulated promoters (e.g., a heat shock promoter)and light-regulated promoters (e.g., a light-inducible promoter or alight-repressible promoter).

Tissue-specific promoters can be, for example, neuron-specificpromoters, glia-specific promoters, muscle cell-specific promoters,heart cell-specific promoters, kidney cell-specific promoters, bonecell-specific promoters, endothelial cell-specific promoters, or immunecell-specific promoters (e.g., a B cell promoter or a T cell promoter).

Developmentally regulated promoters include, for example, promotersactive only during an embryonic stage of development, or only in anadult cell.

In addition to a nucleic acid sequence comprising the disclosed geneticvariants and regulatory sequences, the recombinant expression vectorscan carry additional sequences, such as sequences that regulatereplication of the vector in host cells (e.g., origins of replication)and selectable marker genes. A selectable marker gene can facilitateselection of host cells into which the vector has been introduced (seee.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). For example,a selectable marker gene can confer resistance to drugs, such as G418,hygromycin, or methotrexate, on a host cell into which the vector hasbeen introduced. Exemplary selectable marker genes include, but are notlimited to, the dihydrofolate reductase (DHFR) gene (for use indhfr-host cells with methotrexate selection/amplification), the neo gene(for G418 selection), and the glutamate synthetase (GS) gene.

Additional vectors are described in, for example, U.S. ProvisionalApplication No. 62/367,973, filed on Jul. 28, 2016, which isincorporated herein by reference in its entirety.

The present disclosure also provides compositions comprising any one ormore of the isolated nucleic acid molecules, genomic DNA molecules, cDNAmolecules, or mRNA molecules disclosed herein. In some embodiments, thecomposition is a pharmaceutical composition.

The present disclosure also provides variant GPR156 polypeptides. Thepresent disclosure provides isolated or recombinant polypeptidescomprising at least a portion of the GPR156 protein, wherein the portioncomprises an aspartic acid at the position corresponding to position 533according to SEQ ID NO:4. In some embodiments, the isolated orrecombinant polypeptides comprise at least a portion of the GPR156protein, wherein the portion comprises an aspartic acid at the positioncorresponding to position 529 according to SEQ ID NO:5.

In some embodiments, the isolated or recombinant polypeptide comprisesor consists of an amino acid sequence that has at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% sequence identity to SEQ ID NO:4,wherein the amino acid at the position corresponding to position 533(according to SE ID NO:4) of the polypeptide is an aspartic acid. Insome embodiments, the polypeptide comprises a full-length GPR156 proteincomprising an aspartic acid at the position corresponding to position533 according to SEQ ID NO:4. In some embodiments, the full-lengthGPR156 protein comprises or consists of an amino acid sequence that hasat least about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, or about 99% sequence identity toSEQ ID NO:4, wherein the amino acid at the position corresponding toposition 533 (according to SEQ ID NO:4) of the polypeptide is anaspartic acid. In some embodiments, the polypeptide comprises the aminoacid sequence of SEQ ID NO:4. In some embodiments, the polypeptideconsists of the amino acid sequence of SEQ ID NO:4. The positioncorresponding to Position 516 of this variant GPR156 protein (referringto SEQ ID NO:4) is either a glutamic acid or an aspartic acid. In someembodiments, the position corresponding to Position 516 of this variantGPR156 protein (referring to SEQ ID NO:4) is a glutamic acid. In someembodiments, the position corresponding to Position 516 of this variantGPR156 protein (referring to SEQ ID NO:4) is an aspartic acid.

In some embodiments, the isolated or recombinant polypeptide comprisesor consists of an amino acid sequence that has at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% sequence identity to SEQ ID NO:5,wherein the amino acid at the position corresponding to position 529(according to SEQ ID NO:5) of the polypeptide is an aspartic acid. Insome embodiments, the polypeptide comprises a GPR156 protein comprisingan aspartic acid at the position corresponding to position 529 accordingto SEQ ID NO:5. In some embodiments, the GPR156 protein comprises orconsists of an amino acid sequence that has at least about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, or about 99% sequence identity to SEQ ID NO:5, wherein theamino acid at the position corresponding to position 529 (according toSEQ ID NO:5) of the polypeptide is an aspartic acid. In someembodiments, the polypeptide comprises the amino acid sequence of SEQ IDNO:5. In some embodiments, the polypeptide consists of the amino acidsequence of SEQ ID NO:5. The position corresponding to position 512 ofthis variant GPR156 protein (referring to SEQ ID NO:5) is either aglutamic acid or an aspartic acid. In some embodiments, the positioncorresponding to position 512 of this variant GPR156 protein (referringto SEQ ID NO:5) is a glutamic acid. In some embodiments, the positioncorresponding to position 512 of this variant GPR156 protein (referringto SEQ ID NO:5) is an aspartic acid.

In some embodiments, the polypeptide comprises the portion of the GPR156protein corresponding to positions 310 to 814 of SEQ ID NO:4. In someembodiments, the polypeptide comprises or consists of an amino acidsequence that has at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity to positions 310 to 814 of SEQ ID NO:4,wherein the amino acid at the position (e.g., position 224)corresponding to position 533 according to SEQ ID NO:4 is an asparticacid. In some embodiments, the polypeptide comprises the amino acidsequence of SEQ ID NO:6. In some embodiments, the polypeptide consistsof the amino acid sequence of SEQ ID NO:6. The position corresponding toposition 207 of this GPR156 protein (referring to SEQ ID NO:6) is eithera glutamic acid or an aspartic acid. In some embodiments, the positioncorresponding to position 207 of this GPR156 protein (referring to SEQID NO:6) is a glutamic acid. In some embodiments, the positioncorresponding to position 207 of this GPR156 protein (referring to SEQID NO:6) is an aspartic acid.

The present disclosure also provides fragments of any of thepolypeptides disclosed herein. In some embodiments, the fragmentscomprise at least about 10, at least about 15, at least about 20, atleast about 25, at least about 30, at least about 35, at least about 40,at least about 45, at least about 50, at least about 55, at least about60, at least about 65, at least about 70, at least about 75, at leastabout 80, at least about 85, at least about 90, at least about 95, atleast about 100, at least about 150, at least about 200, at least about250, at least about 300, at least about 350, at least about 400, atleast about 450, at least about 500, at least about 550, at least about600, at least about 650, at least about 700, at least about 750, or atleast about 800 contiguous amino acid residues of the encodedpolypeptide. In this regard, the longer fragments are preferred over theshorter ones. In some embodiments, the fragments comprise at least about10, at least about 15, at least about 20, at least about 25, at leastabout 30, at least about 35, at least about 40, at least about 45, atleast about 50, at least about 55, at least about 60, at least about 65,at least about 70, at least about 75, at least about 80, at least about85, at least about 90, at least about 95, or at least about 100contiguous amino acid residues of the encoded polypeptide. In thisregard, the longer fragments are preferred over the shorter ones.

The present disclosure also provides dimers comprising an isolatedpolypeptide comprising a variant GPR156 wherein the polypeptide isselected from any of the polypeptides disclosed herein.

In some embodiments, the isolated polypeptides disclosed herein arelinked or fused to heterologous polypeptides or heterologous moleculesor labels, numerous examples of which are disclosed elsewhere herein.For example, the proteins can be fused to a heterologous polypeptideproviding increased or decreased stability. The fused domain orheterologous polypeptide can be located at the N-terminus, theC-terminus, or internally within the polypeptide. A fusion partner may,for example, assist in providing T helper epitopes (an immunologicalfusion partner), or may assist in expressing the protein (an expressionenhancer) at higher yields than the native recombinant polypeptide.Certain fusion partners are both immunological and expression enhancingfusion partners. Other fusion partners may be selected to increase thesolubility of the polypeptide or to facilitate targeting the polypeptideto desired intracellular compartments. Some fusion partners includeaffinity tags, which facilitate purification of the polypeptide.

In some embodiments, a fusion protein is directly fused to theheterologous molecule or is linked to the heterologous molecule via alinker, such as a peptide linker. Suitable peptide linker sequences maybe chosen, for example, based on the following factors: 1) the abilityto adopt a flexible extended conformation; 2) the resistance to adopt asecondary structure that could interact with functional epitopes on thefirst and second polypeptides; and 3) the lack of hydrophobic or chargedresidues that might react with the polypeptide functional epitopes. Forexample, peptide linker sequences may contain Gly, Asn and Ser residues.Other near neutral amino acids, such as Thr and Ala may also be used inthe linker sequence. Amino acid sequences which may be usefully employedas linkers include those disclosed in, for example, Maratea et al.,Gene, 1985, 40, 39-46; Murphy et al., Proc. Natl. Acad. Sci. USA, 1986,83, 8258-8262; and U.S. Pat. Nos. 4,935,233 and 4,751,180. A linkersequence may generally be, for example, from 1 to about 50 amino acidsin length. Linker sequences are generally not required when the firstand second polypeptides have non-essential N-terminal amino acid regionsthat can be used to separate the functional domains and prevent stericinterference.

In some embodiments, the polypeptides are operably linked to acell-penetrating domain. For example, the cell-penetrating domain can bederived from the HIV-1 TAT protein, the TLM cell-penetrating motif fromhuman hepatitis B virus, MPG, Pep-1, VP22, a cell-penetrating peptidefrom Herpes simplex virus, or a polyarginine peptide sequence. See,e.g., WO 2014/089290. The cell-penetrating domain can be located at theN-terminus, the C-terminus, or anywhere within the protein.

In some embodiments, the polypeptides are operably linked to aheterologous polypeptide for ease of tracking or purification, such as afluorescent protein, a purification tag, or an epitope tag. Examples offluorescent proteins include, but are not limited to, green fluorescentproteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, AzamiGreen, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellowfluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP,ZsYellowl), blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite,mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g.,eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescentproteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1,DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2,eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent proteins(e.g., mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange,mTangerine, tdTomato), and any other suitable fluorescent protein.Examples of tags include, but are not limited to,glutathione-S-transferase (GST), chitin binding protein (CBP), maltosebinding protein, thioredoxin (TRX), poly(NANP), tandem affinitypurification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG,hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV,KT3, S, S1, T7, V5, VSV-G, histidine (His), biotin carboxyl carrierprotein (BCCP), and calmodulin. In some embodiments, the heterologousmolecule is an immunoglobulin Fc domain, a peptide tag, a transductiondomain, poly(ethylene glycol), polysialic acid, or glycolic acid.

In some embodiments, the isolated polypeptides comprise non-natural ormodified amino acids or peptide analogs. For example, there are numerousD-amino acids or amino acids which have a different functionalsubstituent than the naturally occurring amino acids. The oppositestereo isomers of naturally occurring peptides are disclosed, as well asthe stereo isomers of peptide analogs. These amino acids can readily beincorporated into polypeptide chains by charging tRNA molecules with theamino acid of choice and engineering genetic constructs that utilize,for example, amber codons, to insert the analog amino acid into apeptide chain in a site-specific way.

In some embodiments, the isolated polypeptides are peptide mimetics,which can be produced to resemble peptides, but which are not connectedvia a natural peptide linkage. For example, linkages for amino acids oramino acid analogs include, but are not limited to, —CH₂NH—, —CH₂S—,—CH₂—, —CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CHH₂SO—.Peptide analogs can have more than one atom between the bond atoms, suchas b-alanine, gaminobutyric acid, and the like. Amino acid analogs andpeptide analogs often have enhanced or desirable properties, such as,more economical production, greater chemical stability, enhancedpharmacological properties (half-life, absorption, potency, efficacy,and so forth), altered specificity (e.g., a broad-spectrum of biologicalactivities), reduced antigenicity, and others desirable properties.

In some embodiments, the isolated polypeptides comprise D-amino acids,which can be used to generate more stable peptides because D amino acidsare not recognized by peptidases. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) can be used to generate morestable peptides. Cysteine residues can be used to cyclize or attach twoor more peptides together. This can be beneficial to constrain peptidesinto particular conformations (see, e.g., Rizo and Gierasch, Ann. Rev.Biochem., 1992, 61, 387).

The present disclosure also provides nucleic acid molecules encoding anyof the polypeptides disclosed herein. This includes all degeneratesequences related to a specific polypeptide sequence (all nucleic acidshaving a sequence that encodes one particular polypeptide sequence aswell as all nucleic acids, including degenerate nucleic acids, encodingthe disclosed variants and derivatives of the protein sequences). Thus,while each particular nucleic acid sequence may not be written outherein, each and every sequence is in fact disclosed and describedherein through the disclosed polypeptide sequences.

Percent identity (or percent complementarity) between particularstretches of nucleic acid sequences within nucleic acids or amino acidsequences within polypeptides can be determined routinely using BLASTprograms (basic local alignment search tools) and PowerBLAST programs(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656) or by using the Gap program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, Madison Wis.), using default settings, whichuses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2,482-489). Herein, if reference is made to percent sequence identity, thehigher percentages of sequence identity are preferred over the lowerones.

The present disclosure also provides compositions comprising any one ormore of the nucleic acid molecules and/or any one or more of thepolypeptides disclosed herein and a carrier and/or excipient. In someembodiments, the carrier increases the stability of the nucleic acidmolecule and/or polypeptide (e.g., prolonging the period under givenconditions of storage (e.g., −20° C., 4° C., or ambient temperature) forwhich degradation products remain below a threshold, such as below 0.5%by weight of the starting nucleic acid or protein; or increasing thestability in vivo). Examples of carriers include, but are not limitedto, poly(lactic acid) (PLA) microspheres,poly(D,L-lactic-coglycolic-acid) (PLGA) microspheres, liposomes,micelles, inverse micelles, lipid cochleates, and lipid microtubules. Acarrier may comprise a buffered salt solution such as PBS, HBSS, etc.

The present disclosure also provides methods of producing any of thepolypeptides or fragments thereof disclosed herein. Such polypeptides orfragments thereof can be produced by any suitable method. For example,polypeptides or fragments thereof can be produced from host cellscomprising nucleic acid molecules (e.g., recombinant expression vectors)encoding such polypeptides or fragments thereof. Such methods cancomprise culturing a host cell comprising a nucleic acid molecule (e.g.,recombinant expression vector) encoding a polypeptide or fragmentthereof under conditions sufficient to produce the polypeptide orfragment thereof, thereby producing the polypeptide or fragment thereof.The nucleic acid can be operably linked to a promoter active in the hostcell, and the culturing can be carried out under conditions whereby thenucleic acid is expressed. Such methods can further comprise recoveringthe expressed polypeptide or fragment thereof. The recovering canfurther comprise purifying the polypeptide or fragment thereof.

Examples of suitable systems for protein expression include host cellssuch as, for example: bacterial cell expression systems (e.g.,Escherichia coli, Lactococcus lactis), yeast cell expression systems(e.g., Saccharomyces cerevisiae, Pichia pastoris), insect cellexpression systems (e.g., baculovirus-mediated protein expression), andmammalian cell expression systems.

Examples of nucleic acid molecules encoding polypeptides or fragmentsthereof are disclosed in more detail elsewhere herein. In someembodiments, the nucleic acid molecules are codon optimized forexpression in the host cell. In some embodiments, the nucleic acidmolecules are operably linked to a promoter active in the host cell. Thepromoter can be a heterologous promoter (e.g., a promoter than is not anaturally occurring promoter). Examples of promoters suitable forEscherichia coli include, but are not limited to, arabinose, lac, tac,and T7 promoters. Examples of promoters suitable for Lactococcus lactisinclude, but are not limited to, P170 and nisin promoters. Examples ofpromoters suitable for Saccharomyces cerevisiae include, but are notlimited to, constitutive promoters such as alcohol dehydrogenase (ADHI)or enolase (ENO) promoters or inducible promoters such as PHO, CUP1,GAL1, and G10. Examples of promoters suitable for Pichia pastorisinclude, but are not limited to, the alcohol oxidase I (AOX I) promoter,the glyceraldehyde 3 phosphate dehydrogenase (GAP) promoter, and theglutathione dependent formaldehyde dehydrogenase (FLDI) promoter. Anexample of a promoter suitable for a baculovirus-mediated system is thelate viral strong polyhedrin promoter.

In some embodiments, the nucleic acid molecules encode a tag in framewith the polypeptide or fragment thereof to facilitate proteinpurification. Examples of tags are disclosed elsewhere herein. Such tagscan, for example, bind to a partner ligand (e.g., immobilized on aresin) such that the tagged protein can be isolated from all otherproteins (e.g., host cell proteins). Affinity chromatography, highperformance liquid chromatography (HPLC), and size exclusionchromatography (SEC) are examples of methods that can be used to improvethe purity of the expressed protein.

Other methods can also be used to produce polypeptides or fragmentsthereof. For example, two or more peptides or polypeptides can be linkedtogether by protein chemistry techniques. For example, peptides orpolypeptides can be chemically synthesized using either Fmoc(9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl)chemistry. Such peptides or polypeptides can be synthesized by standardchemical reactions. For example, a peptide or polypeptide can besynthesized and not cleaved from its synthesis resin, whereas the otherfragment of a peptide or protein can be synthesized and subsequentlycleaved from the resin, thereby exposing a terminal group which isfunctionally blocked on the other fragment. By peptide condensationreactions, these two fragments can be covalently joined via a peptidebond at their carboxyl and amino termini, respectively. Alternately, thepeptide or polypeptide can be independently synthesized in vivo asdescribed herein. Once isolated, these independent peptides orpolypeptides may be linked to form a peptide or fragment thereof viasimilar peptide condensation reactions.

In some embodiments, enzymatic ligation of cloned or synthetic peptidesegments allow relatively short peptide fragments to be joined toproduce larger peptide fragments, polypeptides, or whole protein domains(Abrahmsen et al., Biochemistry, 1991, 30, 4151). Alternately, nativechemical ligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method can consist of a two-step chemical reaction (Dawson et al.,Science, 1994, 266, 776-779). The first step can be the chemoselectivereaction of an unprotected synthetic peptide-thioester with anotherunprotected peptide segment containing an amino-terminal Cys residue togive a thioester-linked intermediate as the initial covalent product.Without a change in the reaction conditions, this intermediate canundergo spontaneous, rapid intramolecular reaction to form a nativepeptide bond at the ligation site.

In some embodiments, unprotected peptide segments can be chemicallylinked where the bond formed between the peptide segments as a result ofthe chemical ligation is an unnatural (non-peptide) bond (Schnolzer etal., Science, 1992, 256, 221).

In some embodiments, the polypeptides can possess post-expressionmodifications such as, for example, glycosylations, acetylations, andphosphorylations, as well as other modifications known in the art, bothnaturally occurring and non-naturally occurring. A polypeptide may be anentire protein, or a subsequence thereof.

The present disclosure also provides methods of producing any of thepolypeptides disclosed herein, comprising culturing a host cellcomprising a recombinant expression vectors comprising nucleic acidmolecules comprising a polynucleotide capable of encoding one or more ofthe polypeptides disclosed herein, or its complement, thereby producingthe polypeptide.

The present disclosure also provides cells (e.g., recombinant hostcells) comprising any one or more of the nucleic acid molecules,including vectors comprising the nucleic acid molecules, and/or any oneor more of the polypeptides disclosed herein. The cells can be in vitro,ex vivo, or in vivo. Nucleic acid molecules can be linked to a promoterand other regulatory sequences so they are expressed to produce anencoded protein. Cell lines of such cells are further provided.

In some embodiments, the cell is a totipotent cell or a pluripotent cell(e.g., an embryonic stem (ES) cell such as a rodent ES cell, a mouse EScell, or a rat ES cell). Totipotent cells include undifferentiated cellsthat can give rise to any cell type, and pluripotent cells includeundifferentiated cells that possess the ability to develop into morethan one differentiated cell types. Such pluripotent and/or totipotentcells can be, for example, ES cells or ES-like cells, such as an inducedpluripotent stem (iPS) cells. ES cells include embryo-derived totipotentor pluripotent cells that are capable of contributing to any tissue ofthe developing embryo upon introduction into an embryo. ES cells can bederived from the inner cell mass of a blastocyst and are capable ofdifferentiating into cells of any of the three vertebrate germ layers(endoderm, ectoderm, and mesoderm). In accordance with the presentdisclosure, the embryonic stem cells may be non-human embryonic stemcells.

In some embodiments, the cell is a primary somatic cell, or a cell thatis not a primary somatic cell. Somatic cells can include any cell thatis not a gamete, germ cell, gametocyte, or undifferentiated stem cell.In some embodiments, the cell can also be a primary cell. Primary cellsinclude cells or cultures of cells that have been isolated directly froman organism, organ, or tissue. Primary cells include cells that areneither transformed nor immortal. Primary cells include any cellobtained from an organism, organ, or tissue which was not previouslypassed in tissue culture or has been previously passed in tissue culturebut is incapable of being indefinitely passed in tissue culture. Suchcells can be isolated by conventional techniques and include, forexample, somatic cells, hematopoietic cells, endothelial cells,epithelial cells, fibroblasts, mesenchymal cells, keratinocytes,melanocytes, monocytes, mononuclear cells, adipocytes, preadipocytes,neurons, glial cells, hepatocytes, skeletal myoblasts, and smooth musclecells. For example, primary cells can be derived from connectivetissues, muscle tissues, nervous system tissues, or epithelial tissues.

In some embodiments, the cells may normally not proliferate indefinitelybut, due to mutation or alteration, have evaded normal cellularsenescence and instead can keep undergoing division. Such mutations oralterations can occur naturally or be intentionally induced. Examples ofimmortalized cells include, but are not limited to, Chinese hamsterovary (CHO) cells, human embryonic kidney cells (e.g., HEK 293 cells),and mouse embryonic fibroblast cells (e.g., 3T3 cells). Numerous typesof immortalized cells are well known. Immortalized or primary cellsinclude cells that are typically used for culturing or for expressingrecombinant genes or proteins. In some embodiments, the cell is adifferentiated cell, such as a liver cell (e.g., a human liver cell).

The cell can be from any source. For example, the cell can be aeukaryotic cell, an animal cell, a plant cell, or a fungal (e.g., yeast)cell. Such cells can be fish cells or bird cells, or such cells can bemammalian cells, such as human cells, non-human mammalian cells, rodentcells, mouse cells or rat cells. Mammals include, but are not limitedto, humans, non-human primates, monkeys, apes, cats dogs, horses, bulls,deer, bison, sheep, rodents (e.g., mice, rats, hamsters, guinea pigs),livestock (e.g., bovine species such as cows, steer, etc.; ovine speciessuch as sheep, goats, etc.; and porcine species such as pigs and boars).Birds include, but are not limited to, chickens, turkeys, ostrich,geese, ducks, etc. Domesticated animals and agricultural animals arealso included. The term “non-human animal” excludes humans.

Additional host cells are described in, for example, U.S. ProvisionalApplication No. 62/367,973, filed on Jul. 28, 2016, which isincorporated herein by reference in its entirety.

The nucleic acid molecules and polypeptides disclosed herein can beintroduced into a cell by any means. Transfection protocols as well asprotocols for introducing nucleic acids or proteins into cells may vary.Non-limiting transfection methods include chemical-based transfectionmethods using liposomes, nanoparticles, calcium, dendrimers, andcationic polymers such as DEAE-dextran or polyethylenimine. Non-chemicalmethods include electroporation, sono-poration, and opticaltransfection. Particle-based transfection includes the use of a genegun, or magnet-assisted transfection. Viral methods can also be used fortransfection.

Introduction of nucleic acids or proteins into a cell can also bemediated by electroporation, by intracytoplasmic injection, by viralinfection, by adenovirus, by adeno-associated virus, by lentivirus, byretrovirus, by transfection, by lipid-mediated transfection, or bynucleofection. Nucleofection is an improved electroporation technologythat enables nucleic acid substrates to be delivered not only to thecytoplasm but also through the nuclear membrane and into the nucleus. Inaddition, use of nucleofection in the methods disclosed herein typicallyrequires much fewer cells than regular electroporation (e.g., only about2 million compared with 7 million by regular electroporation). In someembodiments, nucleofection is performed using the LONZA® NUCLEOFECTOR™system.

Introduction of nucleic acids or proteins into a cell can also beaccomplished by microinjection. Microinjection of an mRNA is usuallyinto the cytoplasm (e.g., to deliver mRNA directly to the translationmachinery), while microinjection of a protein or a DNA is usually intothe nucleus. Alternately, microinjection can be carried out by injectioninto both the nucleus and the cytoplasm: a needle can first beintroduced into the nucleus and a first amount can be injected, andwhile removing the needle from the cell a second amount can be injectedinto the cytoplasm. If a nuclease agent protein is injected into thecytoplasm, the protein may comprise a nuclear localization signal toensure delivery to the nucleus/pronucleus.

Other methods for introducing nucleic acid or proteins into a cell caninclude, for example, vector delivery, particle-mediated delivery,exosome-mediated delivery, lipid-nanoparticle-mediated delivery,cell-penetrating-peptide-mediated delivery, orimplantable-device-mediated delivery. Methods of administering nucleicacids or proteins to a subject to modify cells in vivo are disclosedelsewhere herein. Introduction of nucleic acids and proteins into cellscan also be accomplished by hydrodynamic delivery (HDD).

Other methods for introducing nucleic acid or proteins into a cell caninclude, for example, vector delivery, particle-mediated delivery,exosome-mediated delivery, lipid-nanoparticle-mediated delivery,cell-penetrating-peptide-mediated delivery, orimplantable-device-mediated delivery. In some embodiments, a nucleicacid or protein can be introduced into a cell in a carrier such as apoly(lactic acid) (PLA) microsphere, a poly(D,L-lactic-coglycolic-acid)(PLGA) microsphere, a liposome, a micelle, an inverse micelle, a lipidcochleate, or a lipid microtubule.

The present disclosure also provides probes and primers. Examples ofprobes and primers are disclosed above for example. The presentdisclosure provides probes and primers comprising a nucleic acidsequence that specifically hybridizes to any of the nucleic acidmolecules disclosed herein. For example, the probe or primer maycomprise a nucleic acid sequence which hybridizes to a nucleic acidmolecule encoding a GPR156 protein comprising an aspartic acid at theposition corresponding to position at 533 according to SEQ ID NO:4, orwhich hybridizes to the complement of this nucleic acid molecule. Insome embodiments, the probe or primer of the disclosure comprises anucleic acid sequence which hybridizes to a nucleic acid moleculeencoding a GPR156 protein according to SEQ ID NO:4, or which hybridizesto the complement of this nucleic acid molecule. In some embodiments,the probe or primer may comprise a nucleic acid sequence whichhybridizes to a nucleic acid molecule encoding a GPR156 proteincomprising an aspartic acid at the position corresponding to position at529 according to SEQ ID NO:5, or which hybridizes to the complement ofthis nucleic acid molecule. In some embodiments, the probe or primercomprises a nucleic acid sequence which hybridizes to a nucleic acidmolecule encoding a GPR156 protein according to SEQ ID NO:5, or whichhybridizes to the complement of this nucleic acid molecule. The probe orprimer may comprise any suitable length, non-limiting examples of whichinclude at least about 5, at least about 8, at least about 10, at leastabout 11, at least about 12, at least about 13, at least about 14, atleast about 15, at least about 16, at least about 17, at least about 18,at least about 19, at least about 20, at least about 21, at least about22, at least about 23, at least about 24, or at least about 25nucleotides in length. In preferred embodiments, the probe or primercomprises at least about 18 nucleotides in length. The probe or primermay comprise from about 10 to about 35, from about 10 to about 30, fromabout 10 to about 25, from about 12 to about 30, from about 12 to about28, from about 12 to about 24, from about 15 to about 30, from about 15to about 25, from about 18 to about 30, from about 18 to about 25, fromabout 18 to about 24, or from about 18 to about 22 nucleotides inlength. In preferred embodiments, the probe or primer is from about 18to about 30 nucleotides in length.

The present disclosure also provides allele-specific probes andallele-specific primers. The allele-specific probe or primer of thedisclosure specifically hybridizes to a nucleic acid sequence encoding aGPR156 protein comprising an aspartic acid at the position correspondingto position 533 according to SEQ ID NO:4, or to the complement thereof.

In the context of the disclosure “specifically hybridizes” means thatthe probe or primer (e.g. the allele-specific probe or primer) does nothybridize to a nucleic acid sequence encoding a GPR156 protein whichdoes not comprise an aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4. A probe or primer according tothe disclosure preferably encompasses the nucleic acid codon whichencodes the E533D variation n within a GPR156 protein, or the complementthereof.

Thus, the present disclosure provides an allele-specific probe or primercomprising a nucleic acid sequence which is complementary to a nucleicacid sequence encoding a GPR156 protein, wherein the allele-specificprobe or primer comprises a nucleic acid sequence which is complementaryto the nucleic acid codon encoding an aspartic acid at the positioncorresponding to position 533 according to SEQ ID NO:4.

Accordingly, an allele-specific probe or primer according to thedisclosure comprises a nucleic acid sequence which is complementary to anucleic acid sequence encoding the GPR156 protein comprising an asparticacid at position 533 according to SEQ ID NO:4. The allele-specific probeor primer according to the disclosure may be used to detect the E533Dvariation within GPR156 and/or the E529D mutation within the shorterisoform of GPR156. For example, the allele-specific primer according tothe disclosure may be used to amplify the nucleic acid sequence encodingGPR156 (e.g., having the amino acid sequence of SEQ ID NO:4) or afragment thereof comprising the E533D variation. An allele-specificprimer according to the disclosure may also be used to amplify thenucleic acid sequence of a GPR156 isoform (such as the isoform havingthe amino acid sequence of SEQ ID NO: 5) or a fragment thereofcomprising the E529D mutation.

In some embodiments, the allele-specific probe or primer of thedisclosure comprises a nucleic acid sequence which is complementary to anucleic acid sequence encoding a GPR156 protein according to SEQ IDNO:4. In some embodiments, the allele-specific probe or primer comprisesa nucleic acid sequence which is complementary to a nucleic acidsequence encoding a GPR156 protein according to SEQ ID NO:5. In someembodiments, the allele-specific probe or primer comprises a nucleicacid sequence which is complementary to a nucleic acid sequence encodinga GPR156 protein comprising an aspartic acid at the positioncorresponding to position 533 according to SEQ ID NO:4, or to position529 according to SEQ ID NO:5. In some embodiments, the allele-specificprimer specifically hybridizes to any of the nucleic acid moleculesdisclosed herein, wherein the aspartic acid is encoded by the codon GATor GAU. In some embodiments, the allele-specific primer specificallyhybridizes to any of the nucleic acid molecules disclosed herein,wherein the aspartic acid is encoded by the codon GAC. The asparticacid-encoding codon may be at the 3′ end of the allele-specific primer.

In preferred embodiments, the allele-specific probe or primer binds tothe major allele of the GPR156 gene. Thus, the allele-specific primerpreferably binds to the major allele of the GPR156 gene and not to theminor allele of the GPR156 gene. The major allele comprises a nucleicacid sequence encoding an aspartic acid at a position corresponding toposition 516 of SEQ ID NO:4, though the wild-type major allele comprisesa nucleic acid sequence encoding a glutamic acid at a positioncorresponding to position 533 of SEQ ID NO:4. The minor allele comprisesa nucleic acid sequence encoding a glutamic acid at a positioncorresponding to position 516 of SEQ ID NO:4, though the minor allelecomprises a nucleic acid sequence encoding aspartic acid at a positioncorresponding to position 533 of SEQ ID NO:4. Accordingly, theallele-specific probe or primer may specifically hybridize to the minorallele of the GRP156 gene comprising the E533D variation. According tothe disclosure, the allele-specific probe or primer does not hybridizeto the major allele which does not comprise the E533D variation.

The length which is described above with regard to the probe or primerof the disclosure applies, mutatis mutandis, also for theallele-specific probe or primer of the disclosure.

The disclosure also provides a pair of allele-specific primerscomprising one of the allele-specific primers as described above.

In some embodiments, the probe or primer (e.g. the allele-specific probeor primer) comprises DNA. In some embodiments, the probe or primer (e.g.the allele-specific probe or primer) comprises RNA. In some embodiments,the probe (e.g. the allele-specific probe) hybridizes to any of thenucleic acid molecules disclosed herein wherein the aspartic acid (atthe position corresponding to position 533 according to SEQ ID NO:4) isencoded by the codon GAT (or GAU). In some embodiments, the probehybridizes to any of the nucleic acid molecules disclosed herein whereinthe aspartic acid (at the position corresponding to position 533according to SEQ ID NO:4) is encoded by the codon GAC. In someembodiments, the probe (e.g. the allele-specific probe) hybridizes tothe nucleic acid sequence encoding the human GPR156 protein understringent conditions, such as high stringent conditions.

In some embodiments, the probe comprises a label. In some embodiments,the label is a fluorescent label, a radiolabel, or biotin. In someembodiments, the length of the probe is described above. Alternately, insome embodiments, the probe comprises or consists of at least about 20,at least about 25, at least about 30, at least about 35, at least about40, at least about 45, at least about 50, at least about 55, at leastabout 60, at least about 65, at least about 70, at least about 75, atleast about 80, at least about 85, at least about 90, at least about 95,or at least about 100 nucleotides. The probe (e.g., the allele-specificprobe) may be used, for example, to detect any of the nucleic acidmolecules disclosed herein. In preferred embodiments, the probecomprises at least about 18 nucleotides in length. The probe maycomprise from about 10 to about 35, from about 10 to about 30, fromabout 10 to about 25, from about 12 to about 30, from about 12 to about28, from about 12 to about 24, from about 15 to about 30, from about 15to about 25, from about 18 to about 30, from about 18 to about 25, fromabout 18 to about 24, or from about 18 to about 22 nucleotides inlength. In preferred embodiments, the probe is from about 18 to about 30nucleotides in length.

The present disclosure also provides supports comprising a substrate towhich any one or more of the probes disclosed herein is attached. Solidsupports are solid-state substrates or supports with which molecules,such as any of the probes disclosed herein, can be associated. A form ofsolid support is an array. Another form of solid support is an arraydetector. An array detector is a solid support to which multipledifferent probes have been coupled in an array, grid, or other organizedpattern.

Solid-state substrates for use in solid supports can include any solidmaterial to which molecules can be coupled. This includes materials suchas acrylamide, agarose, cellulose, nitrocellulose, glass, polystyrene,polyethylene vinyl acetate, polypropylene, polymethacrylate,polyethylene, polyethylene oxide, polysilicates, polycarbonates, teflon,fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid,polylactic acid, polyorthoesters, polypropylfumerate, collagen,glycosaminoglycans, and polyamino acids. Solid-state substrates can haveany useful form including thin film, membrane, bottles, dishes, fibers,woven fibers, shaped polymers, particles, beads, microparticles, or acombination. Solid-state substrates and solid supports can be porous ornon-porous. A form for a solid-state substrate is a microtiter dish,such as a standard 96-well type. In some embodiments, a multiwell glassslide can be employed that normally contain one array per well. Thisfeature allows for greater control of assay reproducibility, increasedthroughput and sample handling, and ease of automation. In someembodiments, the support is a microarray.

Any of the polypeptides disclosed herein can further have one or moresubstitutions (such as conservative amino acid substitutions),insertions, or deletions that are in addition to the aspartic acid atposition 533 or 529 as described herein. Insertions include, forexample, amino or carboxyl terminal fusions as well as intrasequenceinsertions of single or multiple amino acid residues. Techniques formaking substitutions at predetermined sites in DNA having a knownsequence are well known, for example M13 primer mutagenesis and PCRmutagenesis. Amino acid substitutions are typically of single residues,but can occur at a number of different locations at once; insertionsusually will be on the order of about from 1 to 10 amino acid residues;and deletions will range about from 1 to 30 residues. Deletions orinsertions can be made in adjacent pairs, i.e. a deletion of 2 residuesor insertion of 2 residues. Substitutions, deletions, insertions or anycombination thereof may be combined to arrive at a final construct. Insome embodiments, the mutations do not place the sequence out of readingframe and do not create complementary regions that could producesecondary mRNA structure.

The present disclosure also provides kits for making the compositionsand utilizing the methods described herein. The kits described hereincan comprise an assay or assays for detecting one or more geneticvariants in a sample of a subject.

In some embodiments, the kits for human identification of GPR156variants utilize the compositions and methods described above. In someembodiments, a basic kit can comprise a container having at least onepair of oligonucleotide primers for a locus in any of the nucleic acidmolecules disclosed herein (such as, for example, SEQ ID NOs:9 to 12,SEQ ID NOs:16 to 21, or SEQ ID NOs:25 to 30). A kit can also optionallycomprise instructions for use. A kit can also comprise other optionalkit components, such as, for example, one or more of an allelic ladderdirected to each of the loci amplified, a sufficient quantity of enzymefor amplification, amplification buffer to facilitate the amplification,divalent cation solution to facilitate enzyme activity, dNTPs for strandextension during amplification, loading solution for preparation of theamplified material for electrophoresis, genomic DNA as a templatecontrol, a size marker to insure that materials migrate as anticipatedin the separation medium, and a protocol and manual to educate the userand limit error in use. The amounts of the various reagents in the kitsalso can be varied depending upon a number of factors, such as theoptimum sensitivity of the process. It is within the scope of theseteachings to provide test kits for use in manual applications or testkits for use with automated sample preparation, reaction set-up,detectors or analyzers.

In some embodiments, the kits comprise at least one pair ofoligonucleotide primers for amplification of at least one locus encodingthe aspartic acid, wherein one primer of the pair hybridizes to a SNPnucleobase within a primer binding site of the at least one locus and atleast one primer of the pair comprises a fluorescent label. In someaspects, the locus comprises a variant in the GPR156 gene.

In some embodiments, the kit comprises at least one pair ofoligonucleotide primers for PCR amplification of at least one locusselected from any of the nucleic acid molecules disclosed herein (suchas, for example, SEQ ID NOs:9 to 12, SEQ ID NOs:16 to 21, or SEQ IDNOs:25 to 30), wherein one primer of the pair hybridizes to a SNPnucleobase within a primer binding site of the at least one locus. Insome embodiments, the kit further comprises an allelic laddercorresponding to the at least one locus selected from any of the nucleicacid molecules disclosed herein (such as, for example, SEQ ID NOs:9 to12, SEQ ID NOs:16 to 21, or SEQ ID NOs:25 to 30), at least one of aprotocol, an enzyme, dNTPs, a buffer, a salt or salts, and a controlnucleic acid sample. In some embodiments, the SNP nucleobase is within asequence selected from any of the nucleic acid molecules disclosedherein (such as, for example, SEQ ID NOs:9 to 12, SEQ ID NOs:16 to 21,or SEQ ID NOs:25 to 30).

In some aspects, the kits disclosed herein can comprise a primercomprising a 3′ terminal nucleotide that hybridizes directly to athymine or uracil at nucleotide position 1599 of any of the cDNA or mRNAmolecules disclosed herein (such as, for example, SEQ ID NOs:16 to 21,or SEQ ID NOs:25 to 30).

Also disclosed herein are kits comprising: at least one nucleic acidprobe designed to detect a nucleotide variance in any of the nucleicacid molecules disclosed herein at a position encoding an aspartic acidat the position corresponding to position 533 of SEQ ID NO:4, whereindetection is based on specific hybridization to the nucleotide variancesequence, wherein the nucleic acid probe comprises a detectable label,products and reagents required to carry out an annealing reaction; andinstructions. The kit may comprise any of the probes described herein.

Disclosed herein are kits comprising: at least one pair ofoligonucleotide primers for amplification of at least one locus of anyof the nucleic acid molecules disclosed herein at a position encoding anaspartic acid at the position corresponding to position 533 of SEQ IDNO:4, wherein one primer of the pair hybridizes to a SNP nucleobasewithin a primer binding site of the at least one locus and at least oneprimer of the pair comprises a fluorescent label. The kit may compriseany of the primers described herein.

Disclosed herein are kits comprising at least one pair ofoligonucleotide primers for amplification of at least one locus of anyof the nucleic acid molecules disclosed herein at a position encoding anaspartic acid at the position corresponding to position 533 of SEQ IDNO:4, wherein one primer of the pair hybridizes to a SNP nucleobasewithin a primer binding site of the at least one locus and at least oneprimer of the pair comprises a fluorescent label further comprising anallelic ladder corresponding to the at least one locus of any of thenucleic acid molecules disclosed herein at a position encoding anaspartic acid corresponding to position 533 of SEQ ID NO:4. The kit maycomprise any of the primers described herein.

Disclosed herein are kits comprising at least one pair ofoligonucleotide primers for amplification of at least one locus of anyof the nucleic acid molecules disclosed herein at a position encoding anaspartic acid corresponding to position 533 of SEQ ID NO:4, wherein oneprimer of the pair hybridizes to a SNP nucleobase within a primerbinding site of the at least one locus and at least one primer of thepair comprises a fluorescent label, wherein the primers are used in apolymerase chain reaction (PCR). The kit may comprise any of the primersdescribed herein.

Disclosed herein are kits comprising at least one pair ofoligonucleotide primers for amplification of at least one locus of anyof the nucleic acid molecules disclosed herein at a position encoding anaspartic acid at the position corresponding to position 533 of SEQ IDNO:4, wherein one primer of the pair hybridizes to a SNP nucleobasewithin a primer binding site of the at least one locus and at least oneprimer of the pair comprises a fluorescent label, further comprising atleast one of a protocol, an enzyme, dNTPs, a buffer, a salt or salts,and a control nucleic acid sample. The kit may comprise any of theprimers described herein.

TABLE 1 Primers SEQ SEQ ID ID Forward Primer (5′→3′) NO:Reverse Primer (5′→3′) NO TCTCCCCAG 31 AGAGGGGTC 32 TCTCCCCAT 33AGAGGGGA 34 CCAGAGTCTCCTACCAGATAG 35 AGAAGGCTGGCGAAGAAAGG 36CAACGCCAAAGAGAAGATTG 37 CTTCCAGGTTTGCTGATGACA 38 AAGATATCTGACTCAAAAGAC39 AGAGCCAGGAAAGTGTGAGC 40 TTTCAGAATGATCCTGGCATG 41ACTGTTCTTCCAGGTTTGCTG 42

Those in the art understand that the detection techniques employed aregenerally not limiting. Rather, a wide variety of detection means arewithin the scope of the disclosed methods and kits, provided that theyallow the presence or absence of an amplicon to be determined.

In some aspects, a kit can comprise one or more of the primers or probesdisclosed herein. For example, a kit can comprise one or more probesthat hybridize to one or more of the disclosed genetic variants.

In some aspects, a kit can comprise one of the disclosed cells or celllines. In some aspects, a kit can comprise the materials necessary tocreate a transgenic cell or cell line. For example, in some aspects akit can comprise a cell and a vector comprising a nucleic acid sequencecomprising one or more of the disclosed genetic variants. A kit canfurther comprise media for cell culture.

The present disclosure also provides methods for detecting the presenceof a GPR156 variant gene, mRNA, cDNA, and/or polypeptide in a biologicalsample from a subject human. It is understood that gene sequences withina population and mRNAs and proteins encoded by such genes can vary dueto polymorphisms such as single-nucleotide polymorphisms. The sequencesprovided herein for the GPR156 gene, mRNA, cDNA, and polypeptide areonly exemplary sequences. Other sequences for the GPR156 gene, mRNA,cDNA, and polypeptide are also possible.

The biological sample can be derived from any cell, tissue, orbiological fluid from the subject. The sample may comprise anyclinically relevant tissue, such as a bone marrow sample, a tumorbiopsy, a fine needle aspirate, or a sample of bodily fluid, such asblood, gingival crevicular fluid, plasma, serum, lymph, ascitic fluid,cystic fluid, or urine. In some cases, the sample comprises a buccalswab. The sample used in the methods disclosed herein will vary based onthe assay format, nature of the detection method, and the tissues,cells, or extracts that are used as the sample. A biological sample canbe processed differently depending on the assay being employed. Forexample, when detecting a variant GPR156 nucleic acid molecule,preliminary processing designed to isolate or enrich the sample for thegenomic DNA can be employed. A variety of known techniques may be usedfor this purpose. When detecting the level of variant GPR156 mRNA,different techniques can be used enrich the biological sample with mRNA.Various methods to detect the presence or level of a mRNA or thepresence of a particular variant genomic DNA locus can be used.

In some embodiments, the disclosure provides methods of detecting thepresence or absence of a variant GPR156 nucleic acid molecule comprisingsequencing at least a portion of a nucleic acid in a biological sampleto determine whether the nucleic acid comprises a nucleic acid sequenceencoding an aspartic acid at the position corresponding to position 533according to SEQ ID NO:4 or at the position corresponding to position529 according to SEQ ID NO:5. Any of the variant nucleic acid moleculesdisclosed herein can be detected using any of the probes describedherein.

In some embodiments, the methods of detecting the presence or absence ofa unipolar depression-associated variant GPR156 nucleic acid molecule(e.g., gene, mRNA, or cDNA) in a subject, comprising: performing anassay on a biological sample obtained from the subject, which assaydetermines whether a nucleic acid molecule in the biological samplecomprises a nucleic acid sequence that encodes an aspartic acid at theposition corresponding to position 533 according to SEQ ID NO:4 or atthe position corresponding to position 529 according to SEQ ID NO:5. Insome embodiments, the biological sample comprises a cell or cell lysate.Such methods can further comprise, for example, obtaining a biologicalsample from the subject comprising a GPR156 gene or mRNA, and if mRNA,optionally reverse transcribing the mRNA into cDNA, and performing anassay on the biological sample that determines that a position of theGPR156 gene, mRNA, or cDNA encodes an aspartic acid at the positioncorresponding to position 533 of the variant GPR156 polypeptide. Suchassays can comprise, for example determining the identity of thesepositions of the particular GPR156 nucleic acid molecule. In someembodiments, the subject is a human.

In some embodiments, the methods of detecting the presence or absence ofan anxiety disorder-associated variant GPR156 nucleic acid molecule(e.g., gene, mRNA, or cDNA) in a subject, comprising: performing anassay on a biological sample obtained from the subject, which assaydetermines whether a nucleic acid molecule in the biological samplecomprises a nucleic acid sequence that encodes an aspartic acid at theposition corresponding to position 533 according to SEQ ID NO:4 or atthe position corresponding to position 529 according to SEQ ID NO:5. Insome embodiments, the biological sample comprises a cell or cell lysate.Such methods can further comprise, for example, obtaining a biologicalsample from the subject comprising a GPR156 gene or mRNA, and if mRNA,optionally reverse transcribing the mRNA into cDNA, and performing anassay on the biological sample that determines that a position of theGPR156 gene, mRNA, or cDNA encodes an aspartic acid at the positioncorresponding to position 533 of the variant GPR156 polypeptide. Suchassays can comprise, for example determining the identity of thesepositions of the particular GPR156 nucleic acid molecule. In someembodiments, the subject is a human.

In some embodiments, the assay comprises: sequencing at least a portionof the GPR156 genomic sequence of a nucleic acid molecule in thebiological sample from the subject, wherein the portion sequencedincludes the position corresponding to the position encoding an asparticacid at position 533 in the GPR156 protein according to SEQ ID NO:4, orat the position corresponding to position 529 according to SEQ ID NO:5;sequencing a portion of the GPR156 mRNA sequence of a nucleic acidmolecule in the biological sample from the subject, wherein the portionsequenced includes the position corresponding to the position encodingan aspartic acid at position 533 in the GPR156 protein according to SEQID NO:4, or at the position corresponding to position 529 according toSEQ ID NO:5; or sequencing a portion of the GPR156 cDNA sequence of anucleic acid molecule obtained from mRNA obtained from the biologicalsample obtained from the subject, wherein the portion sequenced includesthe position corresponding to the position encoding an aspartic acid atposition 533 in the GPR156 protein according to SEQ ID NO:4, or at theposition corresponding to position 529 according to SEQ ID NO:5.

In some embodiments, the assay comprises: a) contacting the biologicalsample with a primer hybridizing to: i) a portion of the GPR156 genomicsequence that is proximate to a position of the GPR156 genomic sequenceat the position corresponding to the position encoding an aspartic acidat position 533 according to SEQ ID NO:4, or at the positioncorresponding to position 529 according to SEQ ID NO:5; ii) a portion ofthe GPR156 mRNA sequence that is proximate to a position of the GPR156mRNA corresponding to the position encoding an aspartic acid at position533 according to SEQ ID NO:4, or at the position corresponding toposition 529 according to SEQ ID NO:5; or iii) a portion of the GPR156cDNA sequence that is proximate to a position of the GPR156 cDNAcorresponding to the position encoding an aspartic acid at position 533according to SEQ ID NO:4, or at the position corresponding to position529 according to SEQ ID NO:5; b) extending the primer at least through:i) the position of the GPR156 genomic sequence corresponding tonucleotide positions beyond the codon encoding an aspartic acid atposition 533 according to SEQ ID NO:4 or at the position correspondingto position 529 according to SEQ ID NO:5; ii) the position of the GPR156mRNA corresponding to nucleotide positions beyond the codon encoding anaspartic acid at the position corresponding to position 533 according toSEQ ID NO:4, or at the position corresponding to position 529 accordingto SEQ ID NO:5; or iii) the position of the GPR156 cDNA corresponding tonucleotide positions beyond the codon encoding an aspartic acid at theposition corresponding to position 533 according to SEQ ID NO:4, or atthe position corresponding to position 529 according to SEQ ID NO:5; andc) determining whether the extension product of the primer comprisesnucleotides encoding an aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4, or at the position correspondingto position 529 according to SEQ ID NO:5. In some embodiments, onlyGPR156 genomic DNA is analyzed. In some embodiments, only GPR156 mRNA isanalyzed. In some embodiments, only GPR156 cDNA obtained from GPR156mRNA is analyzed.

In some embodiments, the assay comprises: a) contacting the biologicalsample with an allele-specific primer hybridizing to i) a portion of theGPR156 genomic sequence including the nucleotides encoding an amino acidat the position corresponding to position 533 according to SEQ ID NO:4,or at the position corresponding to position 529 according to SEQ IDNO:5; ii) a portion of the GPR156 mRNA sequence including thenucleotides encoding an amino acid at the position corresponding toposition 533 according to SEQ ID NO:4, or at the position correspondingto position 529 according to SEQ ID NO:5; or iii) a portion of theGPR156 cDNA sequence including the nucleotides encoding an amino acid atthe position corresponding to position 533 according to SEQ ID NO:4, orat the position corresponding to position 529 according to SEQ ID NO:5;b) extending the primer using an allele-specific polymerase chainreaction technique; and c) determining whether extension occurred.Allele-specific polymerase chain reaction techniques can be used todetect mutations such as SNPs in a nucleic acid sequence.Allele-specific primers are used because the DNA polymerase will notextend when a mismatch with the template is present. A number ofvariations of the basic allele-specific polymerase chain reactiontechnique are at the disposal of the skilled artisan.

The allele-specific primer may comprise a nucleic acid sequence which iscomplementary to a nucleic acid sequence encoding the GPR156 proteincomprising an aspartic acid at the position corresponding to position533 according to SEQ ID NO:4, or at the position corresponding toposition 529 according to SEQ ID NO:5, or the complement to the nucleicacid sequence. For example, the allele-specific primer may comprise anucleic acid sequence which is complementary to the nucleic acidsequence encoding SEQ ID NO:4 and/or SEQ ID NO:5, or to the complementto this nucleic acid sequence. The allele-specific primer preferablyspecifically hybridizes to the nucleic acid sequence encoding the GPR156protein when the nucleic acid sequence encodes an aspartic acid at theposition corresponding to position 533 according to SEQ ID NO:4, or atthe position corresponding to position 529 according to SEQ ID NO:5 viathe codon GAT (or GAU) or GAC.

In some embodiments, the assay comprises contacting the biologicalsample with a primer or probe that specifically hybridizes to a variantGPR156 genomic sequence, mRNA sequence, or cDNA sequence and not thecorresponding wild type GPR156 sequence under stringent conditions, anddetermining whether hybridization has occurred.

In some embodiments, the assay comprises RNA sequencing (RNA-Seq). Insome embodiments, the assays also comprise reverse transcribing mRNAinto cDNA via the reverse transcriptase polymerase chain reaction(RT-PCR).

In some embodiments, the methods utilize probes and primers ofsufficient nucleotide length to bind to the target nucleic acid sequenceand specifically detect and/or identify a polynucleotide comprising avariant GPR156 gene, mRNA, or cDNA. The hybridization conditions orreaction conditions can be determined by the operator to achieve thisresult. This nucleotide length may be any length that is sufficient foruse in a detection method of choice, including any assay described orexemplified herein. Generally, for example, primers or probes havingabout 8, about 10, about 11, about 12, about 14, about 15, about 16,about 18, about 20, about 22, about 24, about 26, about 28, about 30,about 40, about 50, about 75, about 100, about 200, about 300, about400, about 500, about 600, or about 700 nucleotides, or more, or fromabout 11 to about 20, from about 20 to about 30, from about 30 to about40, from about 40 to about 50, from about 50 to about 100, from about100 to about 200, from about 200 to about 300, from about 300 to about400, from about 400 to about 500, from about 500 to about 600, fromabout 600 to about 700, or from about 700 to about 800, or morenucleotides in length are used. In preferred embodiments, the probe orprimer comprises at least about 18 nucleotides in length. The probe orprimer may comprise from about 10 to about 35, from about 10 to about30, from about 10 to about 25, from about 12 to about 30, from about 12to about 28, from about 12 to about 24, from about 15 to about 30, fromabout 15 to about 25, from about 18 to about 30, from about 18 to about25, from about 18 to about 24, or from about 18 to about 22 nucleotidesin length. In preferred embodiments, the probe or primer is from about18 to about 30 nucleotides in length.

Such probes and primers can hybridize specifically to a target sequenceunder high stringency hybridization conditions. Probes and primers mayhave complete nucleic acid sequence identity of contiguous nucleotideswith the target sequence, although probes differing from the targetnucleic acid sequence and that retain the ability to specifically detectand/or identify a target nucleic acid sequence may be designed byconventional methods. Accordingly, probes and primers can share about80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequenceidentity or complementarity to the target nucleic acid molecule.

In some embodiments, specific primers can be used to amplify the variantGPR156 locus and/or GPR156 variant mRNA or cDNA to produce an ampliconthat can be used as a specific probe or can itself be detected foridentifying the variant GPR156 locus or for determining the level ofspecific GPR156 mRNA or cDNA in a biological sample. The GPR156 variantlocus can be used to denote a genomic nucleic acid sequence includingpositions corresponding to positions encoding an aspartic acid atposition 533 according to SEQ ID NO:4 or at position 529 according toSEQ ID NO:5. When the probe is hybridized with a nucleic acid moleculein a biological sample under conditions that allow for the binding ofthe probe to the nucleic acid molecule, this binding can be detected andallow for an indication of the presence of the variant GPR156 locus orthe presence or the level of variant GPR156 mRNA or cDNA in thebiological sample. Such identification of a bound probe has beendescribed. The specific probe may comprise a sequence of at least about80%, from about 80% to about 85%, from about 85% to about 90%, fromabout 90% to about 95%, and from about 95% to about 100% identical (orcomplementary) to a specific region of a variant GPR156 gene. Thespecific probe may comprise a sequence of at least about 80%, from about80% to about 85%, from about 85% to about 90%, from about 90% to about95%, and from about 95% to about 100% identical (or complementary) to aspecific region of a variant GPR156 mRNA. The specific probe maycomprise a sequence of at least about 80%, from about 80% to about 85%,from about 85% to about 90%, from about 90% to about 95%, and from about95% to about 100% identical (or complementary) to a specific region of avariant GPR156 cDNA.

In some embodiments, to determine whether the nucleic acid complement ofa biological sample comprises a nucleic acid sequence encoding theaspartic acid at the position corresponding to position 533 according toSEQ ID NO:4, or at the position corresponding to position 529 accordingto SEQ ID NO:5, the biological sample may be subjected to a nucleic acidamplification method using a primer pair that includes a first primerderived from the 5′ flanking sequence adjacent to positions encoding theaspartic acid at the position corresponding to position 533 according toSEQ ID NO:4, or at the position corresponding to position 529 accordingto SEQ ID NO:5 and a second primer derived from the 3′ flanking sequenceadjacent to positions encoding the aspartic acid at the positioncorresponding to position 533 according to SEQ ID NO:4, or at theposition corresponding to position 529 according to SEQ ID NO:5 toproduce an amplicon that is diagnostic for the presence of the SNP atpositions encoding the aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4, or at the position correspondingto position 529 according to SEQ ID NO:5. In some embodiments, theamplicon may range in length from the combined length of the primerpairs plus one nucleotide base pair to any length of amplicon producibleby a DNA amplification protocol. This distance can range from onenucleotide base pair up to the limits of the amplification reaction, orabout twenty thousand nucleotide base pairs. Optionally, the primer pairflanks a region including positions encoding the aspartic acid atposition 533 according to SEQ ID NO:4 or at position 529 according toSEQ ID NO:5 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morenucleotides on each side of positions encoding the aspartic acid atposition 533 according to SEQ ID NO:4 or at position 529 according toSEQ ID NO:5. Similar amplicons can be generated from the mRNA and/orcDNA sequences.

Representative methods for preparing and using probes and primers aredescribed, for example, in Molecular Cloning: A Laboratory Manual, 2ndEd., Vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. 1989 (hereinafter, “Sambrook et al., 1989”);Current Protocols in Molecular Biology, ed. Ausubel et al., GreenePublishing and Wiley-Interscience, New York, 1992 (with periodicupdates) (hereinafter, “Ausubel et al., 1992”); and Innis et al., PCRProtocols: A Guide to Methods and Applications, Academic Press: SanDiego, 1990). PCR primer pairs can be derived from a known sequence, forexample, by using computer programs intended for that purpose, such asthe PCR primer analysis tool in Vector NTI version 10 (Informax Inc.,Bethesda Md.); PrimerSelect (DNASTAR Inc., Madison, Wis.); and Primer3(Version 0.4.0.COPYRGT., 1991, Whitehead Institute for BiomedicalResearch, Cambridge, Mass.). Additionally, the sequence can be visuallyscanned and primers manually identified using known guidelines.

Any nucleic acid hybridization or amplification or sequencing method canbe used to specifically detect the presence of the variant GPR156 genelocus and/or the level of variant GPR156 mRNA or cDNA produced frommRNA. In some embodiments, the nucleic acid molecule can be used eitheras a primer to amplify a region of the GPR156 nucleic acid or thenucleic acid molecule can be used as a probe that specificallyhybridizes, for example, under stringent conditions, to a nucleic acidmolecule comprising the variant GPR156 gene locus or a nucleic acidmolecule comprising a variant GPR156 mRNA or cDNA produced from mRNA.

A variety of techniques are available in the art including, for example,nucleic acid sequencing, nucleic acid hybridization, and nucleic acidamplification. Illustrative examples of nucleic acid sequencingtechniques include, but are not limited to, chain terminator (Sanger)sequencing and dye terminator sequencing.

Other methods involve nucleic acid hybridization methods other thansequencing, including using labeled primers or probes directed againstpurified DNA, amplified DNA, and fixed cell preparations (fluorescencein situ hybridization (FISH)). In some methods, a target nucleic acidmay be amplified prior to or simultaneous with detection. Illustrativeexamples of nucleic acid amplification techniques include, but are notlimited to, polymerase chain reaction (PCR), ligase chain reaction(LCR), strand displacement amplification (SDA), and nucleic acidsequence based amplification (NASBA). Other methods include, but are notlimited to, ligase chain reaction, strand displacement amplification,and thermophilic SDA (tSDA).

Any method can be used for detecting either the non-amplified oramplified polynucleotides including, for example, HybridizationProtection Assay (HPA), quantitative evaluation of the amplificationprocess in real-time, and determining the quantity of target sequenceinitially present in a sample, but which is not based on a real-timeamplification.

Also provided are methods for identifying nucleic acids which do notnecessarily require sequence amplification and are based on, forexample, the known methods of Southern (DNA:DNA) blot hybridizations, insitu hybridization (ISH), and fluorescence in situ hybridization (FISH)of chromosomal material. Southern blotting can be used to detectspecific nucleic acid sequences. In such methods, nucleic acid that isextracted from a sample is fragmented, electrophoretically separated ona matrix gel, and transferred to a membrane filter. The filter boundnucleic acid is subject to hybridization with a labeled probecomplementary to the sequence of interest. Hybridized probe bound to thefilter is detected. In any such methods, the process can includehybridization using any of the probes described or exemplified herein.

In hybridization techniques, stringent conditions can be employed suchthat a probe or primer will specifically hybridize to its target. Insome embodiments, a polynucleotide primer or probe under stringentconditions will hybridize to its target sequence (e.g., the variant(e.g., E533D or E529D) GPR156 gene locus, variant (e.g., E533D or E529D)GPR156 mRNA, or variant (e.g., E533D or E529D) GPR156 cDNA) to adetectably greater degree than to other sequences (e.g., thecorresponding wild type GPR156 locus, wild type mRNA, or wild typecDNA), such as, at least 2-fold, at least 3-fold, at least 4-fold, ormore over background, including over 10-fold over background. In someembodiments, a polynucleotide primer or probe under stringent conditionswill hybridize to its target sequence to a detectably greater degreethan to other sequences by at least 2-fold. In some embodiments, apolynucleotide primer or probe under stringent conditions will hybridizeto its target sequence to a detectably greater degree than to othersequences by at least 3-fold. In some embodiments, a polynucleotideprimer or probe under stringent conditions will hybridize to its targetsequence to a detectably greater degree than to other sequences by atleast 4-fold. In some embodiments, a polynucleotide primer or probeunder stringent conditions will hybridize to its target sequence to adetectably greater degree than to other sequences by over 10-fold overbackground. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. By controlling the stringency ofthe hybridization and/or washing conditions, target sequences that are100% complementary to the probe can be identified (homologous probing).Alternately, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of identity are detected(heterologous probing).

Appropriate stringency conditions which promote DNA hybridization, forexample, 6× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2×SSC at 50° C., are known or can be found inCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. Typically, stringent conditions for hybridization anddetection will be those in which the salt concentration is less thanabout 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration(or other salts) at pH 7.0 to 8.3 and the temperature is at least about30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about60° C. for longer probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. Exemplary low stringency conditions includehybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl,1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1× to 2×SSC(20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplarymoderate stringency conditions include hybridization in 40 to 45%formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at55 to 60° C. Exemplary high stringency conditions include hybridizationin 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about1% SDS. Duration of hybridization is generally less than about 24 hours,usually about 4 to about 12 hours. The duration of the wash time will beat least a length of time sufficient to reach equilibrium.

In hybridization reactions, specificity is typically the function ofpost-hybridization washes, the critical factors being the ionic strengthand temperature of the final wash solution. For DNA-DNA hybrids, theT_(m) can be approximated from the equation of Meinkoth and Wahl, Anal.Biochem., 1984, 138, 267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in the DNA,% form is the percentage of formamide in the hybridization solution, andL is the length of the hybrid in base pairs. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of acomplementary target sequence hybridizes to a perfectly matched probe.T_(m) is reduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization, and/or wash conditions can be adjusted to hybridize tosequences of the desired identity. For example, if sequences with 90%identity are sought, the T_(m) can be decreased 10° C. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1° C.,2° C., 3° C., or 4° C. lower than the thermal melting point (T_(m));moderately stringent conditions can utilize a hybridization and/or washat 6° C., 7° C., 8° C., 9° C., or 10° C. lower than the thermal meltingpoint (T_(m)); low stringency conditions can utilize a hybridizationand/or wash at 11° C., 12° C., 13° C., 14° C., 15° C., or 20° C. lowerthan the thermal melting point Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is optimal to increase the SSCconcentration so that a higher temperature can be used.

Also provided are methods for detecting the presence or quantifying thelevels of variant GPR156 polypeptide in a biological sample, including,for example, protein sequencing and immunoassays. In some embodiments,the method of detecting the presence of GPR156 Glu533Asp (SEQ D NO:4 orSEQ ID NO:5, for example) in a human subject comprises performing anassay on a biological sample from the human subject that determines thepresence of GPR156 Glu533Asp (SEQ ID NO:4 or SEQ ID NO:5, for example)in the biological sample.

Illustrative non-limiting examples of protein sequencing techniquesinclude, but are not limited to, mass spectrometry and Edmandegradation. Illustrative examples of immunoassays include, but are notlimited to, immunoprecipitation, Western blot, immunohistochemistry,ELISA, immunocytochemistry, flow cytometry, and immuno-PCR. Polyclonalor monoclonal antibodies detectably labeled using various knowntechniques (e.g., calorimetric, fluorescent, chemiluminescent, orradioactive) are suitable for use in the immunoassays.

The present disclosure also provides methods for diagnosing unipolardepression or detecting a risk of unipolar depression in a humansubject, comprising: detecting an alteration in a nucleic acid moleculeencoding a GPR156 protein obtained from the human subject, wherein thealteration encodes an aspartic acid at the position corresponding toposition 533 in the GPR156 protein according to SEQ ID NO:4, or at theposition corresponding to position 529 according to SEQ ID NO:5; anddiagnosing the human subject with unipolar depression if the subject hasone or more symptoms of depression, including one or more symptoms ofunipolar depression, or diagnosing the human subject as at risk forunipolar depression if the subject does not have one or more symptoms ofdepression. In some embodiments, the human subject is in need of suchdiagnosis. In some embodiments, the human subject may have relativesthat have been diagnosed with unipolar depression. The alteration maycomprise a change from a G to a T (or U) or a C at the positioncorresponding to position 1599 according to SEQ ID NO:13. In someembodiments, the alteration changes the sequence of the codon encodingthe position corresponding to position 533 in the GPR156 protein to GAT(or GAU). In some embodiments, the alteration changes the sequence ofthe codon encoding the position corresponding to position 533 in theGPR156 protein to GAC.

The Diagnostic and Statistical Manual of Mental Disorders—Fifth Edition(DSM-V), published by the American Psychiatric Association, WashingtonD.C., 2013, is the primary diagnostic reference of Mental Healthprofessionals in the United States. According to the DSM-V, unipolardepression consists of: major depressive disorder, dysthymic disorder,mixed depressive disorder, adjustment disorder with depressed mood, anddepression not otherwise specified (NOS). DSMIV criteria for depressionis for 2 weeks or more, five or more symptoms from the following: i)feeling depressed mood most of the day nearly every day; ii) markedlydiminished interest or pleasure in all or almost all activities; iii)significant weight loss or decreased appetite; iv) insomnia orhypersomnia; v) psychomotor agitation or retardation; vi) fatigue orloss of energy; vii) feelings of worthlessness or excessive guilt; viii)diminished ability to think or concentrate or indecisiveness; and ix)recurrent thoughts of death. DSMIV criteria for dysthymic disorder ischaracterized by an overwhelming yet chronic state of depression,exhibited by a depressed mood for most of the days, for more days thannot, for at least 2 years. The person who suffers from this disordermust not have gone for more than 2 months without experiencing two ormore of the following symptoms: i) poor appetite or overeating; ii)insomnia or hypersomnia; iii) low energy or fatigue; iv) lowself-esteem; v) poor concentration or difficulty making decisions; andvi) feelings of hopelessness.

The present disclosure also provides methods for diagnosing an anxietydisorder or detecting a risk of an anxiety disorder in a human subject,comprising: detecting an alteration in a nucleic acid molecule encodinga GPR156 protein obtained from the human subject, wherein the alterationencodes an aspartic acid at the position corresponding to position 533in the GPR156 protein according to SEQ ID NO:4, or at the positioncorresponding to position 529 according to SEQ ID NO:5; and diagnosingthe human subject with an anxiety disorder if the subject has one ormore symptoms of an anxiety disorder, including one or more symptoms ofclinical neuropsychiatric anxiety disorder, or diagnosing the humansubject as at risk for an anxiety disorder if the subject does not haveone or more symptoms of an anxiety disorder. In some embodiments, thehuman subject is in need of such diagnosis. In some embodiments, thehuman subject may have relatives that have been diagnosed with ananxiety disorder. The alteration may comprise a change from a G to a T(or U) or a C at the position corresponding to position 1599 accordingto SEQ ID NO:13. In some embodiments, the alteration changes thesequence of the codon encoding the position corresponding to position533 in the GPR156 protein to GAT (or GAU). In some embodiments, thealteration changes the sequence of the codon encoding the positioncorresponding to position 533 in the GPR156 protein to GAC. In someembodiments, the anxiety disorder is clinical neuropsychiatric anxietydisorder.

Physiological symptoms of an anxiety disorder include, but are notlimited to, muscle tension, heart palpitations, sweating, dizziness, andshortness of breath. Emotional symptoms include, but are not limited to,restlessness, a sense of impending doom, fear of dying, fear ofembarrassment or humiliation, and fear of something terrible happening.

In some embodiments, the methods comprise detecting the presence of thevariant GPR156 genomic DNA, mRNA, or cDNA obtained from mRNA obtainedfrom a biological sample obtained from the subject. It is understoodthat gene sequences within a population and mRNAs encoded by such genescan vary due to polymorphisms such as single nucleotide polymorphisms(SNPs). The sequences provided herein for the GPR156 gene, mRNA, cDNA,and polypeptide are only exemplary sequences and other such sequences,including GPR156 alleles other than the alleles encoding aspartic acidat the positions corresponding to positions 533 (according to SEQ IDNO:4)/529 (according to SEQ I NO:5) in the GPR156 protein, are alsopossible.

In some embodiments, the detecting step comprises sequencing at least aportion of the nucleic acid molecule that encodes a GPR156 protein,wherein the sequenced nucleic acid molecule encodes an amino acidsequence which comprises the position corresponding to position 533according to SEQ ID NO:4 or position 529 according to SEQ ID NO:5 of theGPR156 protein. Any of the nucleic acid molecules disclosed herein(e.g., genomic DNA, mRNA, or cDNA) can be sequenced. In someembodiments, the detecting step comprises sequencing the entire nucleicacid molecule.

In some embodiments, the detecting step comprises: amplifying at least aportion of the nucleic acid molecule that encodes a GPR156 protein,wherein the amplified nucleic acid molecule encodes an amino acidsequence which comprises the position corresponding to position 533according to SEQ ID NO:4 (and/or to position 529 according to SEQ IDNO:5); labeling the nucleic acid molecule with a detectable label;contacting the labeled nucleic acid with a support comprising a probe,wherein the probe comprises a nucleic acid sequence which hybridizesunder stringent conditions to a nucleic acid sequence encoding asparticacid at the position corresponding to position at 533 according to SEQID NO:4 (and/or to position 529 according to SEQ ID NO:5); and detectingthe detectable label. Any of the nucleic acid molecules disclosed hereincan be amplified. For example, any of the genomic DNA, cDNA, or mRNAmolecules disclosed herein can be amplified. In some embodiments, thenucleic acid molecule is mRNA and the method further comprisesreverse-transcribing the mRNA into a cDNA prior to the amplifying step.

In some embodiments, the detecting step comprises: contacting thenucleic acid molecule with a probe comprising a detectable label,wherein the probe comprises a nucleic acid sequence which hybridizesunder stringent conditions to a nucleic acid sequence encoding an aminoacid sequence which comprises aspartic acid at the positioncorresponding to position 533 according to SEQ ID NO:4 (and/or toposition 529 according to SEQ ID NO:5), and detecting the detectablelabel. In some embodiments, the nucleic acid molecule is present withina cell obtained from the human subject, such that the detection isaccording to an in situ hybridization technique.

In some embodiments, the detecting step comprises contacting the nucleicacid molecule with an allele-specific primer, and amplifying the nucleicacid molecule using allele-specific PCR techniques. The allele-specificprimer may be any such primer described herein, and may be specific toalleles of GPR156 that encode an aspartic acid at the positioncorresponding to position 533 according to SEQ ID NO:4 and/or position529 according to SEQ ID NO:5.

Other assays that can be used in the methods disclosed herein include,for example, reverse transcription polymerase chain reaction (RT-PCR) orquantitative RT-PCR (qRT-PCR). Yet other assays that can be used in themethods disclosed herein include, for example, RNA sequencing (RNA-Seq)followed by determination of the presence and quantity of variant mRNAor cDNA in the biological sample.

The present disclosure also provides methods for identifying a humansubject having unipolar depression or a risk for developing unipolardepression. The methods generally comprise determining in a sampleobtained from the subject the presence or absence of an E533D variationwithin the GPR156 protein; and/or the presence or absence of a nucleicacid molecule comprising a mutation encoding an E533D variation withinthe GPR156 protein. The presence of the E533D variation within theGPR156 protein and/or presence of a nucleic acid molecule encoding anE533D variation indicates that the subject has unipolar depression or arisk for developing unipolar depression. The method may be carried outin vitro, in situ, or in vivo.

The present disclosure also provides methods for identifying a humansubject having an anxiety disorder or a risk for developing an anxietydisorder. The methods generally comprise determining in a sampleobtained from the subject the presence or absence of an E533D variationwithin the GPR156 protein; and/or the presence or absence of a nucleicacid molecule comprising a mutation encoding an E533D variation withinthe GPR156 protein. The presence of the E533D variation within theGPR156 protein and/or presence of a nucleic acid molecule encoding anE533D variation indicates that the subject has an anxiety disorder or arisk for developing an anxiety disorder. The method may be carried outin vitro, in situ, or in vivo. In some embodiments, the anxiety disorderis clinical neuropsychiatric anxiety disorder.

In some embodiments of the method, the determining step comprisessequencing at least a portion of the nucleic acid molecule that encodesa GPR156 protein. The sequenced nucleic acid molecule may encode anamino acid sequence which comprises a position corresponding to position533 according to SEQ ID NO:4. The determining step may comprisesequencing the nucleic acid molecule encoding the entire GPR156 protein.In some embodiments of the method, the determining step comprisesamplifying at least a portion of the nucleic acid molecule that encodesa GPR156 protein, labeling the nucleic acid molecule with a detectablelabel, contacting the labeled nucleic acid with a support comprising aprobe, wherein the probe comprises a nucleic acid sequence whichspecifically hybridizes, including, for example, under stringentconditions, to a nucleic acid sequence encoding aspartic acid at theposition corresponding to position 533 according to SEQ ID NO:4, anddetecting the detectable label. The amplified nucleic acid moleculepreferably encodes an amino acid sequence which comprises the positioncorresponding to position 533 according to SEQ ID NO:4. If the nucleicacid includes mRNA, the method may further comprise reverse-transcribingthe mRNA into a cDNA prior to the amplifying step. In some embodiments,the determining step comprises contacting the nucleic acid molecule witha probe comprising a detectable label and detecting the detectablelabel. The probe preferably comprises a nucleic acid sequence whichspecifically hybridizes, including, for example, under stringentconditions, to a nucleic acid sequence encoding an amino acid sequencewhich comprises aspartic acid at the position corresponding to positionat 533 according to SEQ ID NO:4. The nucleic acid molecule may bepresent within a cell obtained from the human subject. As part of themethod, the E533D variation within the GPR156 protein may be encoded bythe nucleic acid sequence GAT, or may be encoded by the nucleic acidsequence GAC.

The present disclosure also provides methods for diagnosing unipolardepression or detecting a risk of unipolar depression in a humansubject, comprising: detecting a variant GPR156 protein, such as aprotein comprising SEQ ID NO:4 or SEQ ID NO:5, obtained from the humansubject; and diagnosing the human subject with unipolar depression ifthe subject has one or more symptoms of depression, or diagnosing thehuman subject as at risk for unipolar depression if the subject does nothave one or more symptoms of depression. In some embodiments, the humansubject is in need of such diagnosis. In some embodiments, the humansubject may have relatives that have been diagnosed with unipolardepression. In some embodiments, the depression or unipolar depressionis not childhood depression and/or anxiety.

The present disclosure also provides methods for diagnosing an anxietydisorder or detecting a risk of an anxiety disorder in a human subject,comprising: detecting a variant GPR156 protein, such as a proteincomprising SEQ ID NO:4 or SEQ ID NO:5, obtained from the human subject;and diagnosing the human subject with an anxiety disorder if the subjecthas one or more symptoms of an anxiety disorder, or diagnosing the humansubject as at risk for an anxiety disorder if the subject does not haveone or more symptoms of an anxiety disorder. In some embodiments, thehuman subject is in need of such diagnosis. In some embodiments, thehuman subject may have relatives that have been diagnosed with ananxiety disorder. In some embodiments, the anxiety disorder is clinicalneuropsychiatric anxiety disorder.

In some embodiments, the method further comprises treating the subjectwith an antidepressant when the alteration is detected in the subjectand the subject is diagnosed as having unipolar depression.

In some embodiments, the antidepressant is a selective serotoninreuptake inhibitor (SSRI). Suitable examples of SSRIs include, but arenot limited to, Citalopram (Celexa®), Escitalopram (Lexapro®,Cipralex®), Paroxetine (Paxil®, Seroxat®), Fluoxetine (Prozac®),Fluvoxamine (Luvox®), and Sertraline (Zoloft®, Lustral®).

In some embodiments, the antidepressant is a serotonin-norepinephrinereuptake inhibitor (SNRA). Suitable examples of SNRIs include, but arenot limited to, Desvenlafaxine (Pristiq®), Duloxetine (Cymbalta®),Levomilnacipran (Fetzima®), Milnacipran (Ixel®, Savella®), Tofenacen(Elamol®, Tofacine®), and Venlafaxine (Effexor®).

In some embodiments, the antidepressant is a norepinephrine reuptakeinhibitor (NRI). Suitable examples of NRIs include, but are not limitedto, Reboxetine (Edronax®), Viloxazine (Vivalan®), and Atomoxetine(Strattera).

In some embodiments, the antidepressant is lithium.

In some embodiments, the antidepressant is a serotonin modulator andstimulator (SMS). Suitable examples of SMSs include, but are not limitedto, Vilazodone (Viibryd®) and Vortioxetine (Trintellix®).

In some embodiments, the antidepressant is a serotonin antagonist andreuptake inhibitor (SARI). Suitable examples of SARIs include, but arenot limited to, Etoperidone (Axiomin®, Etonin®), Nefazodone (Nefadar®,Serzone®), and Trazodone (Desyrel®).

In some embodiments, the antidepressant is a tricyclic antidepressant(TCA). Suitable examples of TCAs include, but are not limited to,Amitriptyline (Elavil®, Endep®), Amitriptylinoxide (Amioxid®,Ambivalon®, Equilibrin®), Clomipramine (Anafranil®), Desipramine(Norpramin®, Pertofrane®), Dibenzepin (Noveril®, Victoril®),Dimetacrine) (Istonil®), Dosulepin (Prothiaden®), Doxepin (Adapin®,Sinequan®), Imipramine (Tofranil®), Lofepramine (Lomont®, Gamanil®),Melitracen (Dixeran®, Melixeran®, Trausabun®), Nitroxazepine(Sintamil®), Nortriptyline (Pamelor®, Aventyl®), Noxiptiline (Agedal®,Elronon®, Nogedal®), Pipofezine (Azafen®/Azaphen®), Protriptyline(Vivactil®), and Trimipramine (Surmontil®). Also included areButriptyline (Evadyne®), demexiptiline (Deparon®, Tinoran®),imipraminoxide (Imiprex®, Elepsin®), iprindole (Prondol®, Galatur®,Tetran®), metapramine (Timaxel®), propizepine (Depressin®, Vagran®), andquinupramine (Kinupril®, Kevopril®). Also included are Opipramol(Insidon®) and Tianeptine (Stablon®).

In some embodiments, the antidepressant is a tetracyclic antidepressants(TeCA). Suitable examples of TeCAs include, but are not limited to,Amoxapine (Asendin®), Maprotiline (Ludiomil®), Mianserin (Bolvidon®,Norval®, Tolvon®), Mirtazapine (Remeron®), and Setiptiline (Tecipul®).

In some embodiments, the antidepressant is a monoamine oxidaseinhibitors (MAOI). Suitable examples of MAOIs include, but are notlimited to, Iproniazid (Marsilid®), Isocarboxazid (Marplan®), Phenelzine(Nardil®), Selegiline (Eldepryl®, Zelapar®, Emsam®), Tranylcypromine(Parnate®), Metralindole (Inkazan®), Moclobemide (Aurorix®, Manerix®),Pirlindole (Pirazidol®), and Toloxatone (Humoryl®). Others include, forexample, benmoxin) (Neuralex®), Caroxazone (Surodil®, Timostenil®),iproclozide (Sursum®), mebanazine) (Actomol®), nialamide (Niamid®),octamoxin (Ximaol®), pheniprazine (Catron®), phenoxypropazine(Drazine®), pivhydrazine (Tersavid®), safrazine (Safra®), Eprobemide(Befol®), and minaprine (Brantur®, Cantor®).

In some embodiments, the antidepressant is an atypical antipsychotic.Suitable examples of atypical antipsychotic include, but are not limitedto, Amisulpride (Solian®), Lurasidone (Latuda®), and Quetiapine(Seroquel®).

In some embodiments, the antidepressant is Agomelatine (Valdoxan®),Bifemelane (Alnert®, Celeport®), Bupropion (Wellbutrin®), Ketamine(Ketalar®), Tandospirone (Sediel®), or Teniloxazine (Lucelan®,Metatone®).

In some embodiments, the method further comprises treating the subjectwith an anxiolytic agent when the alteration is detected in the subjectand the subject is diagnosed as having an anxiety disorder. In someembodiments, the anxiety disorder is clinical neuropsychiatric anxietydisorder.

In some embodiments, the anxiolytic agent is a benzodiazepine, includingbut not limited to, Alprazolam (Xanax®), Bromazepam (Lectopam®,Lexotan®), Chlordiazepoxide (Librium®), Clonazepam (Klonopin®,Rivotri®), Clorazepate (Tranxene®), Diazepam (Valium®), Flurazepam(Dalmane®), Lorazepam (Ativan®), Oxazepam (Serax®, Serapax®), Temazepam(Restoril®), Triazolam (Halcion®), and Tofisopam (Emandaxin®,Grandaxin®).

In some embodiments, the anxiolytic agent is a carbamate, including butnot limited to, meprobamate (Miltown®, Equanil®).

In some embodiments, the anxiolytic agent is an antihistamine, includingbut not limited to, Hydroxyzine (Atarax®), Chlorpheniramine(Chlor-Trimeton®), and diphenhydramine (Benadryl®).

In some embodiments, the anxiolytic agent is an azapirone, including butnot limited to, Buspirone (Buspar®) and Tandospirone (Sediel®).

In some embodiments, the anxiolytic agent is an SSRI, SNRI, TCA, TeCA,or MAOI, as described herein.

In some embodiments, the anxiolytic agent is Mebicar (Mebicarum®),Fabomotizole (Afobazole®), Selank, Bromantane, Emoxypine, Pregabalin,Menthyl isovalerate, or Menthyl isovalerate (Validol®).

Administration of the antidepressant or anxiolytic agents can be by anysuitable route including, but not limited to, parenteral, intravenous,oral, subcutaneous, intra-arterial, intracranial, intrathecal,intraperitoneal, topical, intranasal, or intramuscular. Pharmaceuticalcompositions for administration are desirably sterile and substantiallyisotonic and manufactured under GMP conditions. Pharmaceuticalcompositions can be provided in unit dosage form (i.e., the dosage for asingle administration). Pharmaceutical compositions can be formulatedusing one or more physiologically and pharmaceutically acceptablecarriers, diluents, excipients or auxiliaries. The formulation dependson the route of administration chosen. The term “pharmaceuticallyacceptable” means that the carrier, diluent, excipient, or auxiliary iscompatible with the other ingredients of the formulation and notsubstantially deleterious to the recipient thereof.

The present disclosure also provides antidepressants for use in thetreatment of unipolar depression in a human subject comprising analteration in a gene encoding the human GPR156 protein, wherein thealteration encodes an aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4 or to position 529 according toSEQ ID NO:4. The disclosure further provides antidepressants for use inthe manufacture of a medicament for the treatment of unipolar depressionin a human subject comprising an alteration in a gene encoding the humanGPR156 protein, wherein the alteration encodes an aspartic acid at theposition corresponding to position 533 according to SEQ ID NO:4 or toposition 529 according to SEQ ID NO:5 in the human GPR156 protein. Insome embodiments, the alteration changes the sequence of the codonencoding the position corresponding to position 533 according to SEQ IDNO:4 or to position 529 according to SEQ ID NO:5 to GAT (or GAU). Insome embodiments, the alteration changes the sequence of the codonencoding the position corresponding to position 533 according to SEQ IDNO:4 or to position 529 according to SEQ ID NO:5 to GAC. In someembodiments, the antidepressant is a selective serotonin reuptakeinhibitor, a serotonin-norepinephrine reuptake inhibitor, or anorepinephrine reuptake inhibitor. In some embodiments, theantidepressant is lithium.

In some embodiments of the antidepressant for use in the treatment ofunipolar depression in a human subject, the human subject has beentested positive for the E533D variation within the GPR156 protein and/orfor a nucleic acid molecule encoding the E533D variation within theGPR156 protein. In some embodiments, the treatment comprises the step ofdetermining whether or not the human subject has a GPR156 protein withthe E533D variation and/or a nucleic acid molecule encoding a GPR156protein with the E533D variation. In some embodiments, the human subjecthas been identified as having unipolar depression or as having a riskfor developing unipolar depression according to a method for identifyinga human subject having unipolar depression or a risk for developingunipolar depression, including any such method described or exemplifiedherein. The E533D variation refers to a mutation resulting in anaspartic acid at the position corresponding to position 533 according toSEQ ID NO:4. In some embodiments, the E533D variation within the GPR156protein is encoded by the codon GAT. In some embodiments, the E533Dvariation within the GPR156 protein is encoded by the codon GAC. In someembodiments, the antidepressant for use in the treatment of unipolardepression in a human subject is a selective serotonin reuptakeinhibitor, a serotonin-norepinephrine reuptake inhibitor, or anorepinephrine reuptake inhibitor. In some embodiments, theantidepressant for the use in the treatment of unipolar depression in ahuman subject is lithium.

The present disclosure also provides anxiolytic agents for use in thetreatment of an anxiety disorder in a human subject comprising analteration in a gene encoding the human GPR156 protein, wherein thealteration encodes an aspartic acid at the position corresponding toposition 533 according to SEQ ID NO:4 or to position 529 according toSEQ ID NO:4. The disclosure further provides anxiolytic agents for usein the manufacture of a medicament for the treatment of an anxietydisorder in a human subject comprising an alteration in a gene encodingthe human GPR156 protein, wherein the alteration encodes an asparticacid at the position corresponding to position 533 according to SEQ IDNO:4 or to position 529 according to SEQ ID NO:5 in the human GPR156protein. In some embodiments, the alteration changes the sequence of thecodon encoding the position corresponding to position 533 according toSEQ ID NO:4 or to position 529 according to SEQ ID NO:5 to GAT (or GAU).In some embodiments, the alteration changes the sequence of the codonencoding the position corresponding to position 533 according to SEQ IDNO:4 or to position 529 according to SEQ ID NO:5 to GAC. In someembodiments, the anxiolytic agent is a benzodiazepine, a carbamate, anantihistamine, an azapirone, an SSRI, an SNRI, a TCA, a TeCA, or anMAOI. In some embodiments, the anxiety disorder is clinicalneuropsychiatric anxiety disorder.

In some embodiments of the anxiolytic agent for use in the treatment ofan anxiety disorder in a human subject, the human subject has beentested positive for the E533D variation within the GPR156 protein and/orfor a nucleic acid molecule encoding the E533D variation within theGPR156 protein. In some embodiments, the treatment comprises the step ofdetermining whether or not the human subject has a GPR156 protein withthe E533D variation and/or a nucleic acid molecule encoding a GPR156protein with the E533D variation. In some embodiments, the human subjecthas been identified as having an anxiety disorder or as having a riskfor developing an anxiety disorder according to a method for identifyinga human subject having an anxiety disorder or a risk for developing ananxiety disorder, including any such method described or exemplifiedherein. The E533D variation refers to a mutation resulting in anaspartic acid at the position corresponding to position 533 according toSEQ ID NO:4. In some embodiments, the E533D variation within the GPR156protein is encoded by the codon GAT. In some embodiments, the E533Dvariation within the GPR156 protein is encoded by the codon GAC. In someembodiments, the anxiolytic agent for use in the treatment of an anxietydisorder in a human subject is a benzodiazepine, a carbamate, anantihistamine, an azapirone, an SSRI, an SNRI, a TCA, a TeCA, or anMAOI. In some embodiments, the anxiety disorder is clinicalneuropsychiatric anxiety disorder.

The present disclosure also provides uses of any of the variant GPR156genes, mRNAs, cDNAs, polypeptides, and hybridizing nucleic acidmolecules disclosed herein in the diagnosis of unipolar depression ordiagnosing the risk of developing unipolar depression.

The present disclosure also provides uses of any of the variant GPR156genes, mRNAs, cDNAs, polypeptides, and hybridizing nucleic acidmolecules disclosed herein in the diagnosis of an anxiety disorder ordiagnosing the risk of developing an anxiety disorder. In someembodiments, the anxiety disorder is clinical neuropsychiatric anxietydisorder.

All patent documents, websites, other publications, accession numbersand the like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise, if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the present disclosure can be used incombination with any other feature, step, element, embodiment, or aspectunless specifically indicated otherwise. Although the present disclosurehas been described in some detail by way of illustration and example forpurposes of clarity and understanding, it will be apparent that certainchanges and modifications may be practiced within the scope of theappended claims.

The nucleotide and amino acid sequences recited herein are shown usingstandard letter abbreviations for nucleotide bases, and one-letter codefor amino acids. The nucleotide sequences follow the standard conventionof beginning at the 5′ end of the sequence and proceeding forward (i.e.,from left to right in each line) to the 3′ end. Only one strand of eachnucleotide sequence is shown, but the complementary strand is understoodto be included by any reference to the displayed strand. The amino acidsequences follow the standard convention of beginning at the aminoterminus of the sequence and proceeding forward (i.e., from left toright in each line) to the carboxy terminus.

The following examples are provided to describe the embodiments ingreater detail. They are intended to illustrate, not to limit, theclaimed embodiments.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C. or is at ambienttemperature, and pressure is at or near atmospheric.

Example 1: Patient Recruitment and Phenotyping

Whole exome sequencing was performed in a family of Mennonite ancestrythat had been ascertained for genetic evaluation of unipolar depression.Individuals in this pedigree were additionally systematically evaluatedby a neuropsychiatric team using a Structured Clinical Interview forDSM-5 (SCID-5). This semi-structured interview is administered by atrained mental health professional, and is designed to ensure systematicand accurate diagnosis of DSM-5 disorders. That interview was used todetermine if individuals and their relatives meet criteria for MajorDepression, Generalized Anxiety Disorder, and any other comorbidpsychiatric conditions. The interview typically took one to two hours toconduct with the subject. In addition to the interview, collateralinformation was gathered to confirm diagnoses from the subject's medicalrecords, treatment providers, and close contacts with the subject'swritten consent. The exomes of all available affected and unaffectedfamily members in this large pedigree encompassing a total of 50individuals were sequenced.

Example 2: Genomic Samples

Genomic DNA was extracted from peripheral blood samples and transferredto the Regeneron Genetics Center (RGC) for whole exome sequencing, andstored in automated biobanks at −80° C. Fluorescence-basedquantification was performed to ensure appropriate DNA quantity andquality for sequencing purposes.

1 μg of DNA was sheared to an average fragment length of 150 base pairs(Covaris LE220) and prepared for exome capture with a custom reagent kitfrom Kapa Biosystems. Samples were captured using the NimbleGen SeqCapVCRome 2.1 or the Integrated DNA Technologies xGen exome target designs.Samples were barcoded, pooled, and multiplexed for sequenced using 75 bppaired-end sequencing on an Illumina HiSeq 2500 with v4 chemistry.Captured fragments were sequenced to achieve a minimum of 85% of thetarget bases covered at 20× or greater coverage. Following sequencing,data was processed using a cloud-based pipeline developed at the RGCthat uses DNAnexus and AWS to run standard tools for sample-level dataproduction and analysis. Briefly, sequence data were generated andde-multiplexed using Illumina's CASAVA software. Sequence reads weremapped and aligned to the GRCh37/hg19 human genome reference assemblyusing BWA-mem. After alignment, duplicate reads were marked and flaggedusing Picard tools and indels were realigned using GATK to improvevariant call quality. SNP and INDEL variants and genotypes were calledusing GATK's HaplotypeCaller and Variant Quality Score Recalibration(VQSR) from GATK was applied to annotate the overall variant qualityscores. Sequencing and data quality metric statistics were captured foreach sample to evaluate capture performance, alignment performance, andvariant calling.

Example 3: Genomic Data Analyses

Standard quality-control filters for minimum read depth (>10), genotypequality (>30), and allelic balance (>15%) were applied to calledvariants. Passing variants were classified and annotated based on theirpotential functional effects (whether synonymous, nonsynonymous,splicing, frameshift, or nonframeshift variants) using an RGC developedannotation and analysis pipeline. Familial relationships were verifiedthrough identity by descent (IBD) derived metrics from genetic data toinfer relatedness and relationships in the cohort using PRIMUS (Stapleset al., Amer. J. Human Genet., 2014, 95, 553-564) and cross-referencingwith the reported pedigree for this family.

Pedigree-based variant analyses and segregation were performed toidentify candidate disease genes under an autosomal dominant inheritancepattern given the reported family history. Primary analysis wasperformed using as many informative affected individuals that wereavailable for sequencing to reduce potential confounders given byincomplete penetrance of the disorder, age of onset, or potentialexposure to environmental factors that may trigger symptomaticpresentation of the disease. Shared variants among all affectedindividuals were subsequently annotated and filtered by their observedfrequencies in population control databases such as dbSNP, the 1000Genomes Project, the NHLBI Exome Sequencing Project, the ExomeAggregation Consortium Database (ExAc), and internal RGC databases tofilter out common polymorphisms and high frequency, likely benignvariants. Algorithms for bioinformatic prediction of functional effectsof variants, such as LRT, Poly-phen2, SIFT, CADD, and Mutation Taster,along with conservation scores based on multiple species alignments(i.e. GERP, PhastCons, PhyloP) were incorporated as part of theannotation process of variants and used to inform on the potentialdeleteriousness of identified candidate variants.

A single rare variant was identified in the gene GPR156(hg19.g.chr3:119886725(C>A); c.1599G>T; p.Glu533Asp, p.E533D)segregating with the phenotype of unipolar depression in the affectedfamily members of this pedigree. Additional family members not includedin the primary analysis but that also carried the p.E533D variants hadconfirmed diagnoses of anxiety disorder. To exclude other possiblevariants segregating with the depression or anxiety disorder phenotypein this family, single point exome-wide and multi-point genome-widelinkage analyses were performed using a customized RGC developedpipeline based on the published MERLIN algorithm (Abecasis et al., Nat.Genet., 2002, 30). Briefly, parametric linkage analysis of SNP chip andexome derived genotypes was performed using MERLIN(csg.sph.umich.edu/abecasis/merlin/index.html). PLINK formattedgenotypes were restricted to single nucleotide variants (SNVs) presentin at least one non-founder. Allele frequencies were estimated infounders and other unrelated members of the pedigree. Individualswithout genetic data were assigned missing/dummy genotypes. fcGENE(sourceforge.net/projects/fcgene/) was used to convert PLINK formatgenotypes to MERLIN formatted ped files. Additional files (.dat and.map) necessary for linkage analysis were prepared using customizedscripts. The pedigree structure was validated using Pedstats(csg.sph.umich.edu/abecasis/pedstats/index.html). Linkage analysis ofchip data was carried out using either step-wise or grid spacing betweengenotypes with no assumptions about recombination. Single-point linkagewas used to analyze exome based genotypes. For all analyses, anincomplete penetrance model was assumed (0.0001, 0.75, 1) with a diseaseallele frequency of approximately of 0.01%.

For both exome-wide and genome-wide analyses, a single peak of linkagewas identified in this pedigree reaching a maximum LOD score of 2.98 andexpanding a 14.4 Megabase (Mb) region in human chromosome 3. Within thislinkage region the GPR156 gene is located and the candidate variant(c.1599G>T, p.Glu533Asp) identified through the initial rare-variantfamily-based segregation analysis achieved a single marker LOD score of2.56.

As demonstrated herein, these genetic analyses indicate that GPR156 is agene associated with increased familial susceptibility to unipolardepression and anxiety disorder.

Additionally, nucleotide fragments comprising the wild type and mutantE533D sequences were subcloned into a vector (see, FIGS. 1 and 2) toobtain wild type and variant E533D GPR156 constructs for additionalanalyses.

Example 4: Detection

The presence of a certain genetic variant in a subject can indicate thatthe subject has an increased risk of having or developing a unipolardepression. A sample, such as a blood sample, can be obtained from asubject. Nucleic acids can be isolated from the sample using commonnucleic acid extraction kits. After isolating the nucleic acid from thesample obtained from the subject, the nucleic acid is sequenced todetermine if there is a genetic variant present. The sequence of thenucleic acid can be compared to a control sequence (wild type sequence).Finding a difference between the nucleic acid obtained from the sampleobtained from the subject and the control sequence indicates thepresence of a genetic variant. These steps can be performed as describedin the examples above and throughout the present disclosure. Thepresence of one or more genetic variants is indicative of the subject'sincreased risk for having or developing unipolar depression.

Each reference (including, but not limited to, journal articles, U.S.and non-U.S. patents, patent application publications, internationalpatent application publications, gene bank accession numbers, and thelike) cited in the present application is incorporated herein byreference in its entirety.

What is claimed:
 1. A molecular complex comprising single stranded DNAhybridized to an allele-specific primer or an allele-specific probe;wherein the DNA encodes a GPR156 protein comprising an amino acidsequence according to SEQ ID NO:4 or SEQ ID NO:5; and wherein theallele-specific primer or allele-specific probe is hybridized tonucleotides of the DNA that encode an aspartic acid at a positioncorresponding to position 533 according to SEQ ID NO:4 or at a positioncorresponding to position 529 according to SEQ ID NO:5.
 2. The molecularcomplex according to claim 1, wherein the DNA comprises the nucleic acidsequence according to SEQ ID NO:9.
 3. The molecular complex according toclaim 1, wherein the DNA consists of the nucleic acid sequence accordingto SEQ ID NO:9.
 4. The molecular complex according to claim 1, whereinthe DNA comprises the nucleic acid sequence according to SEQ ID NO:10.5. The molecular complex according to claim 1, wherein the DNA consistsof the nucleic acid sequence according to SEQ ID NO:10.
 6. The molecularcomplex according to claim 1, wherein the DNA comprises the nucleic acidsequence according to SEQ ID NO:11.
 7. The molecular complex accordingto claim 1, wherein the DNA consists of the nucleic acid sequenceaccording to SEQ ID NO:11.
 8. The molecular complex according to claim1, wherein the DNA comprises the nucleic, acid sequence according to SEQID NO:12.
 9. The molecular complex according to claim 1, wherein the DNAconsists of the nucleic acid sequence according to SEQ ID NO:12.
 10. Themolecular complex according to claim 1, wherein the allele-specificprimer or allele-specific probe comprises from 0.10 to 35 nucleotides inlength.
 11. The molecular complex according to claim 1, wherein theallele-specific primer or allele-specific probe comprises DNA.
 12. Themolecular complex according to claim 1, wherein the allele-specificprimer or allele-specific probe comprises RNA.
 13. The molecular complexaccording to claim 1, wherein the allele-specific primer orallele-specific probe is detectably labeled.
 14. The molecular complexaccord ng to claim 13, wherein the detectable label is a fluorophore, ahapten, an enzyme, a radiolabel, a pigment, a dye, a chemiluminescentsubstance, or a metal-containing substance.
 15. The molecular complexaccording to claim 1, wherein the allele-specific probe is attached to asupport.
 16. The molecular complex according to claim 15, wherein thesupport is an array.